Hemoglobin-like protein comprising genetically fused globin-like polypeptides

ABSTRACT

The alpha subunits of hemoglobin, which in nature are formed as separate polypeptide chains which bind noncovalently to the beta subunits, are here provided in the form of the novel molecule di-alpha globin, a single polypeptide chain defined by connecting the two alpha subunits either directly via peptide bond or indirectly by a flexible amino acid or peptide linker. Di-alpha globin may be combined in vivo or in vitro with beta globin and heme to form hemoglobin. Di-alpha globin is expressed by recombinant DNA techniques. Di-beta globin may be similarly obtained. 
     We further describe the production of tetrameric human hemoglobin and di-alpha/beta 2  hemoglobin in the yeast Saccharomyces cerevisiae. The synthesis of the protein is directed by a synthetic promotor consisting of two functional parts, an upstream activator sequence (UAS) that confers inducible transcription by galactose from a consensus yeast transcriptional initiation site. The expression construct is designed such that translation is expected to initiate at the same position as the human wild-type genes for α- and β-globin. Three different types of expression vectors have been used: (1) α-globin and β-globin contained on two separate plasmids (pGS4688 and pGS4988) in a diploid yeast strain; (2) α-globin and β-globin each contained on a single plasmid (pGS289 and pGS389) and expressed in either haploid or diploid strains; and (3) di-alpha-globin and beta globin contained on a single plasmid (pGS 3089)and expressed in haploid strains. 
     Finally, we describe the co-expression of alpha and beta globin chains. The chains are folded together and combined intracellularly with heme to form active tetrameric hemoglobin. The hemoglobin may be recovered from the cells&#39; soluble fraction. The invention thus obviates the need to express alpha and beta globin separately, solubilize, renature and purify them, and combine them in vitro with heme to obtain an artificial hemoglobin. By way of comparison, the separately expressed beta globin known in the art is deposited in inclusion bodies. Polycistronic co-expression of alpha and beta globins is particularly preferred.

This is a division of application Ser. No. 07/789,179 filed Nov. 8,1991, now U.S. Pat. No. 5,545,727, which is a CIP of Ser. No. 07/671,707filed Apr. 1, 1991, now abandoned, which is a CIP of PCT/US90/02654filed May 10, 1990, now abandoned, which is a CIP of (a) Ser. No.07/374,161 filed Jun. 30, 1989, now abandoned, (b) Ser. No. 07/379,116filed Jul. 13, 1989, now abandoned, and (c) Ser. No. 07/349,623 filedMay 10, 1989, now abandoned, all hereby incorporated by referenceherein.

CROSS-REFERENCE TO RELATED APPLICATIONS

Hoffman and Nagai, U.S. Ser. No. 07/194,338, filed May 10, 1988, nowU.S. Pat. No. 5,028,588, presently owned by Somatogen, Inc., relates tothe use of low oxygen affinity and other mutant hemoglobins as bloodsubstitutes, and to the expression of alpha and beta globin innonerythroid cells. Hoffman and Nagai, U.S. Ser. No. 07/443,950, filedDec. 1, 1989, discloses certain additional dicysteine hemoglobinmutants; it is a continuation-in-part of Ser. No. 07/194,338. Anderson,et al., HEMOGLOBINS AS DRUG DELIVERY AGENTS Atty. Docket.:ANDERSON5-U.S.A., filed Nov. 8, 1991, discloses use of conjugation ofhemoglobins with drugs as a means for delivery of the drug to a patient.

FIELD OF THE INVENTION

The present invention relates to the intracellular assembly of ahemoglobin-like protein in biologically functional, substantiallysoluble form through co-expression of alpha- and beta-globin-likepolypeptides in bacterial or yeast cells.

It further relates to the genetic cross-linking of the two alphasubunits of hemoglobin to form a novel polypeptide, di-alpha globin,which may be considered a partially assembled intermediate leading to ahemoglobin-like protein, and the use of this compound in the productionof synthetic hemoglobins having an increased intravascular half-life ascompared to stroma-free hemoglobins. It also relates to the analogouspolypeptide di-beta globin.

INFORMATION DISCLOSURE STATEMENT

It is not always practical to transfuse a patient with donated blood. Inthese situations, use of a red blood cell substitute is desirable. Theproduct must effectively transport O₂, just as do red blood cells.("Plasma expanders", such as dextran and albumin, do not transportoxygen.) The two types of substitutes that have been studied mostextensively are hemoglobin solutions and fluorocarbon emulsions.

A. Structure and Function of Hemoglobin

Hemoglobin (Hgb) is the oxygen-carrying component of blood. Hemoglobincirculates through the bloodstream inside small enucleate cells callederythrocytes (red blood cells). Hemoglobin is a protein constructed fromfour associated polypeptide chains, and bearing prosthetic groups knownas hemes. The erythrocyte helps maintain hemoglobin in its reduced,functional form. The heme iron atom is susceptible to oxidation, but maybe reduced again by one of two enzyme systems within the erythrocyte,the cytochrome b₅ and glutathione reduction systems.

About 92% of the normal adult human hemolysate is Hgb A (designatedalpha2 beta2, because it comprises two alpha and two beta chains). Thealpha chain consists of 141 amino acids. The iron atom of the heme(ferroprotoporphyrin IX) group is bound covalently to the imidazole ofHis 87 (the "proximal histidine"). The beta chain is 146 residues longand heme is bound to it at His 92. Apohemoglobin is the heme-freeanalogue of hemoglobin; it exists predominantly as the αβ-globin dimer.

Separated, heme-free, alpha-and beta-globins have been prepared from theheme-containing alpha and beta subunits of hemoglobin. The separatedheme-free globin chains are folded very differently, even though theheme-containing subunits are highly similar in secondary structure andbasic folding features. This shows that the binding of the prostheticheme group to globin subunits has quite different effects on alpha andbeta globin. Yip, et al., J. Biol. Chem., 247: 7237-44 (1972).

Native human hemoglobin has been fully reconstituted from separatedheme-free alpha globin and beta globin and hemin. Preferably, heme isfirst added to the alpha-globin subunit. The heme-bound alpha globin isthen complexed to the heme-free beta subunit. Finally, heme is added tothe half-filled globin dimer, and tetrameric hemoglobin is obtained.Yip, et al., PNAS (U.S.A.), 74: 64-68 (1977).

In cell-free systems prepared from unfractionated rabbit reticulocytehemolysates, globin is actively synthesized for approximately fiveminutes, and then protein synthesis abruptly ceases. Prior addition ofhemin prevents or delays the cessation of synthetic activity, as aresult of the effect of hemin on an inhibitory protein known as"hemin-regulated inhibitor" (HRI). Hemin deficiency has a more severeeffect on alpha chain synthesis than on beta chain synthesis as alphaglobin mRNA is less efficient than beta-globin mRNA in initiatingpolypeptide chain synthesis. It has been speculated that alpha chainsare released from their site of synthesis only in the presence of freebeta chains, which immediately complex the released alpha chains to formαβ-globin dimers. These then combine with heme to form tetramerichemoglobin. Winterhalter and Huehns, J. Biol. Chem., 239: 3699 (1964).It is certainly known that the addition of heme to αβ-globin dimers(apohemoglobin) leads to the rapid formation of hemoglobin.

The human alpha and beta globin genes reside on chromosomes 16 and 11,respectively. Bunn and Forget, Hemoglobin: Molecular, Genetic andclinical Aspects (W. B. Saunders Co., Philadelphia Pa. 1986). Both geneshave been cloned and sequenced, Liebhaber, et al., PNAS 77: 7054-58(1980) (alpha-globin genomic DNA); Marotta, et al., J. Biol. Chem., 252:5040-53 (1977) (beta globin cDNA); Lawn, et al., Cell, 21:647 (1980)(beta globin genomic DNA).

Hemoglobin exhibits cooperative binding of oxygen by the four subunitsof the hemoglobin molecule (two alpha-globins and two beta-globins inthe case of Hgb A), and this cooperativity greatly facilitates efficientoxygen transport. Cooperativity, achieved by the so-called heme-hemeinteraction, allows hemoglobin to vary its affinity for oxygen.Hemoglobin reversibly binds up to four moles of oxygen per mole of Hgb.

Oxygen-carrying compounds are frequently compared by means of a deviceknown as an oxygen dissociation curve. This curve is obtained when, fora given oxygen carrier, oxygen saturation or content is graphed againstthe partial pressure of oxygen. For Hgb, the percentage of saturationincreases with partial pressure according to a sigmoid relationship. TheP₅₀ is the partial pressure at which the oxygen-carrying solution ishalf saturated with oxygen. It is thus a measure of oxygen-bindingaffinity; the higher the P₅₀, the more loosely the oxygen is held.

When the oxygen dissociation curve of an oxygen-carrying solution issuch that the P₅₀ is less than that for whole blood, it is said to be"left-shifted."

The oxygen affinity of hemoglobin is lowered by the presence of2,3-diphosphoglycerate (2,3-DPG), chloride ions and hydrogen ions.Respiring tissue releases carbon dioxide into the blood and lowers itspH (i.e. increases the hydrogen ion concentration), thereby causingoxygen to dissociate from hemoglobin and allowing it to diffuse intoindividual cells.

The ability of hemoglobin to alter its oxygen affinity, increasing theefficiency of oxygen transport around the body, is dependent on thepresence of the metabolite 2,3-DPG. Inside the erythrocyte 2,3-DPG ispresent at a concentration nearly as great as that of hemoglobin itself.In the absence of 2,3-DPG "conventional" hemoglobin binds oxygen verytightly and would release little oxygen to respiring tissue.

Aging erythrocytes release small amounts of free hemoglobin into theblood plasma where it is rapidly bound by the scavenging proteinhaptoglobin. The hemoglobin-haptoglobin complex is removed from theblood and degraded by the spleen and liver.

B. Blood Substitutes, Generally

It is clear from the above considerations that free native hemoglobin A,injected directly into the bloodstream, would not support efficientoxygen transport about the body. The essential allosteric regulator2,3-DPG is not present in sufficient concentration in the plasma toallow hemoglobin to release much oxygen at venous oxygen tension.

Nonetheless, solutions of conventional hemoglobin have been used as RBCsubstitutes. The classic method of preparing hemoglobin solutionsemploys outdated blood. The red cells are lysed and cellular debris isremoved, leaving what is hopefully "stromal-free hemoglobin" (SFH).

Several basic problems have been observed with this approach. Thesolution must be freed of any toxic components of the red cell membranewithout resorting to cumbersome and tedious procedures which woulddiscourage large-scale production. DeVenuto, "Appraisal of HemoglobinSolution as a Blood Substitute", Surgery Gynecology and Obstetrics, 149:417-436 (1979).

Second, as expected, such solutions are "left-shifted" (lower P₅₀) ascompared to whole blood. Gould, et al., "The Development of PolymerizedPyridoxylated Hemoglobin Solution as a Red Cell Substitute", Ann. Emerg.Med. 15: 1416-1419 (Dec. 3, 1986). As a result, the oxygen affinity istoo high to unload enough oxygen into the tissues. Benesch and Benesch,Biochem. Biophys. Res. Comm., 26:162-167 (1967).

Third, SFH has only a limited half-life in the circulatory system. Thisis because oxy Hgb partially dissociates into a dimer (αβ) that israpidly cleared from the blood by glomerular filtration and binding tocirculating haptoglobulin. If large amounts of soluble hemoglobin areintroduced into the circulation, glomerular filtration of the dimers maylead to a protein and iron load on the kidney capable of causing renaldamage. Bunn, H. F., et al. (1969) The renal handling of hemoglobin I.Glomerular filtration. J. Exp. Med. 129:909-923; Bunn, H. F., and J. H.Jandl; (1969) The renal handling of hemoglobin II. Catabolism. J. Exp.Med. 129:925-934; Lee, R. L., et al. (1989) Ultrapure, stroma-free,polymerized bovine hemoglobin solution: Evaluation of renal toxicity(blood substitutes) J. Surgical Res. 47:407-411; Feola, M., et al.(1990) Nephrotoxicity of hemoglobin solutions. Biomat. Art. Cell Art.Org., 18(2):233-249; Tam, S. C. and J. T. F. Wong (1988) Impairment ofrenal function by stroma-free hemoglobin in rats. J. Lab. Clin. Med.111:189-193.

Finally, SFH has a high colloid osmotic pressure (COD). Thus,administration of SFH in a dose that would have the same oxygen-carryingcapacity as a unit of packed red blood cells is inadvisable, since thehigh osmotic pressure (60 mm Hg) would cause a massive influx of waterfrom the cells into the bloodstream, thus dehydrating the patient'stissues. This consideration limits the dose of SFH to that which providea final concentration of about 6-8 gm Hgb/dl.

In an effort to restore the desired P₅₀, researchers added 2,3-DPG tothe hemoglobin solution. Unfortunately, 2,3-DPG was rapidly eliminatedfrom the circulation. Scientists then turned to other organicphosphates, particularly pyridoxal phosphate. Like 2,3-DPG, thesecompounds stabilized the "T state" of the Hgb by forming a salt bridgebetween the N-termini of the two beta chains. The pyridoxylatedhemoglobin had a P₅₀ of 20-22 torr, as compared to 10 torr for SFH and28 torr for whole blood. While this is an improvement over SFH, thepyridoxylated Hgb remains "high affinity" relative to whole blood.

C. Chemical Crosslinking of Hemoglobin Subunits

The properties of hemoglobin have been altered by specificallychemically crosslinking the alpha chains between the Lys99 of alpha1 andthe Lys99 of alpha2. Walder, U.S. Pat. Nos. 4,600,531 and 4,598,064;Snyder, et al., PNAS (U.S.A.) 84: 7280-84 (1987); Chaterjee, et al., J.Biol. Chem., 261: 9927-37 (1986). The P₅₀ was 29 mm Hg, and renalexcretion was abrogated by the crosslinking, but the plasma half-lifewas increased just 2-3 fold.

This chemical crosslinking was accomplished by reactingbis(3,5-dibromosalicyl)fumarate with deoxyhemoglobin A in the presenceof inositol hexaphosphate. This reaction has a low yield (10-20%).Moreover, purification is required to eliminate derivatives modified atother sites (there are 42 other lysine residues and the amino terminalamino groups of the four chains at which competing reactions couldoccur).

A further problem with the use of a "diaspirin" crosslinking agent isthat it can participate in a side reaction yielding a carcinogenichalophenol.

In the hemoglobin analogue of the present invention, the N-terminalvaline and C-terminal arginine of the alpha globins are connected bymeans of an amino acid or peptide linker, without resort of specialcoupling agents.

The beta chains have also been chemically crosslinked. Kavanaugh, etal., Biochemistry, 27: 1804-8(1988). Kavanaugh notes that the betaN-termini are 16 Å apart in the T state and 20 Å apart in the R state.Not surprisingly, the introduction of a DIDS bridge between theN-termini of T state hemoglobin hindered the shift to the R state,thereby decreasing the O₂ affinity of the molecule. While the Kavanaughanalogue has desirable oxygen binding and renal clearancecharacteristics, it too is obtained in low yield.

D. Gene Expression, Generally

Gene expression embraces the transcription of DNA into messenger RNA,and the translation of messenger RNA into protein. The process oftranscription begins when an enzyme, DNA-directed RNA polymerase, bindsto DNA. The binding site for this enzyme is often called the "promoter,"and the binding of the enzyme to the promoter may be controlled byvarious repressors or inducers of transcription. The RNA polymeraseslides along the DNA molecule, manufacturing a messenger RNA transcript.When it encounters a second regulatory element, the "terminator," theenzyme falls off, and the mRNA transcript is formed.

Messenger RNA is used by the ribosomes, the protein factories of thecell, as a template for the construction of the corresponding protein.The ribosomal binding site comprises the so-called Shine Delgarno (SD)sequence and a properly spaced initiation (start) codon. Beginning at aspecial RNA triplet known as the initiation codon, transfer RNAs bind tocorresponding codons of the messenger. Each transfer RNA is two-handed;it binds to the messenger codon by means of a complementary anti-codon,while holding the corresponding amino acid in position to be linked intothe growing polypeptide chain. The chain falls off when the ribosomeencounters one of three special triplets known as "stop" codons. Thatpart of the original gene which corresponds to the messenger sequencefrom the initiator codon to the last codon before the stop codon isknown as the coding sequence. There is also a 5'-flanking sequence,which begins with the promoter, and a 3'-flanking sequence which endswith the terminator.

E. Polycistronic Expression

It is possible for a single messenger RNA transcript to have onepromoter, but two or more pairs of start and stop codons that definedistinctly translatable sequences. Each such sequence is known as a"cistron," and the polypeptide corresponding to the cistrons are thusco-expressed under the control of the single promoter.

The majority of bacterial operons are polycistronic, that is, severaldifferent genes are transcribed as a single message from their operons.Examples include the lactose operon with three linked genes (lacZ, lacYand lacA) and the tryptophan operon with five associated genes (trpE,trpD, trpC, trpB, and trpA). In these operons, the synthesis ofmessenger RNA is initiated at the promoter and, within the transcript,coding regions are separated by intercistronic regions of variouslengths. (An operon is a cluster of genes that is controlled as a singletranscriptional genetic unit). Translational efficiency varies fromcistron to cistron. Kastelein, et al., Gene, 23: 245-54 (1983).

When intercistronic regions are longer than the span of the ribosome(about 35 bases), dissociation at the stop codon of one cistron isfollowed by independent initiation at the next cistron. With shorterintercistronic regions, or with overlapping cistrons, the 30S subunit ofa terminating ribosome may fail to dissociate from the polycistronicmRNA, being instantly attracted to the next translational initiationsite. Lewin, Gene Expression, 143-148 (John Wiley & Sons: 1977).

Unlike bacterial mRNAs, eukaroyotic mRNAs are generally monocistronic innature. Lewin, Gene Expression, 157.

Synthetic polycistronic operons have been constructed and expressed inboth prokaryotes and eukaryotes.

Lee, et al., Nucleic Acids Res., 12: 6797 (1984) describe a special caseof a synthetic polycistronic operon in which all of the cistrons expressthe same polypeptide. This homopolycistronic structure was constructedto maximize the gene dosage effect.

Schoner, et al., PNAS, 83: 8506-10 (1986) translated a synthetictwo-cistron mRNA in E. coli. The first cistron was a short, arbitraryAU-rich sequence, while the second cistron was a mammalian gene (bGH).It was found that "read through" translation occurred if the stop codonof the first cistron followed the SD element of the second cistron andlay close to the start codon of the second cistron. Schoner's purposewas to overcome his failure to express Met-bGH with its native codons athigh levels, possibly as a result of inhibition of translation by localsecondary structures. The first cistron was engineered to favor ribosomebinding (by placing the SD sequence and the AUG initiation codon in anAU-rich region free of local secondary structure). See also Schoner, etal., Meth. Enzymol., 153: 401-416 (1987); which reveals that bGHoverproduction by this technique was associated with the formation ofprotein granules.

Saito, et al., J. Biochem., 101: 1281-88 (1987) expressed a syntheticsomatomedin C gene in E. coli using a two cistron system. They theorizedthat the instability of somatomedin C, a basic polypeptide, might beovercome by complexing it with an acidic polypeptide. Thus, theyconstructed a two-cistron system which could express both polypeptides.The termination codon of the first cistron overlapped the initiationcodon of the second cistron. The transformants accumulated Somatomedin Cat high levels. However, the somatomedin C was recovered in the form ofinsoluble pellets (see page 1282).

The ribosomes of mammalian cells are likewise capable of reinitiatingtranslation at an initiation codon downstream from a termination codon.Thus, Boel, et al., FEBS Lett., 219:181 (1987) expressed a dicistronictranscription unit in mammalian (CHO) cells. This unit directedsynthesis of both the precursor of human pancreatic polypeptide and of aselectable genetic marker (mouse DHFR).

CODON, WO88/05486 describes the production of dicistronic mRNA whichencodes both a protein of interest (e.g., tissue plasminogen activator)and a selectable phenotype (e.g., neomycin resistance). The commonpromoter was, in each of the examples a derivative of the Harvey murinesarcoma virus, and the dicistronic mRNA was translated in suitableeukaryotic cells.

GENENTECH, EP Appl 117,058 discloses the expression in vertebrate hostcells of a dicistronic expression vector wherein one cistron codes forthe desired protein (e.g., HbsAg) and a second codes for a secondprotein (e.g., DHFR) whose synthesis is subject to environmental control(e.g., with methotrexate).

F. Fused Genes and Proteins, Generally

Genes may be fused together by removing the stop codon of the firstgene, and joining it in phase to the second gene. Parts of genes mayalso be fused, and spacer DNAs which maintain phase may be interposedbetween the fused sequences. The product of a fused gene is a singlepolypeptide, not a plurality of polypeptides as is expressed by apolycistronic operon. Different genes have been fused together for avariety of purposes. Thus, Gilbert, U.S. Pat. No. 4,338,397 inserted arat preproinsulin gene behind a fragment of the E. coli penicillinasegene. His purpose was to direct E. coli transformants to secrete theexpression product of the fused gene. Fused genes have also beenprepared so that a non-antigenic polypeptide may be expressed alreadyconjugated to an immunogenic carrier protein. The present invention,however, contemplates the joining of two copies of the same gene.

The use of linker DNA sequences to join two different DNA sequences isknown. These linkers are used to provide restriction sites for DNAcleavage, or to encode peptides having a unique character thatfacilitates purification of the encoded fusion protein or a fragmentthereof. See, e.g., Rutter, U.S. Pat. No. 4,769,326.

The lectin of Pisum sativum seeds is synthesized as a single 275-aminoacid preproprotein consisting of a signal sequence followed first by thebeta chain and then by the alpha chain. The signal sequence is removedin the endoplasmic reticulum, and in the protein bodies the resulting"prolectin" is cleaved into a 187-AA beta chain and a 58-AA alpha chain.(Further processing results in truncation at the carboxyl termini).While the pea seed isolate is thus a heterodimer, it was discovered thatthe uncleaved naturally-occurring "prolectin" also binds carbohydrates,and that this "prolectin" could be expressed in E. coli and recovered infunctional form. Stubbs, et al., J. Biol. Chem., 261: 6141-44 (1986).

Toth, U.S. Pat. No. 4,774,180 teaches the expression of polyprotein.This polyprotein was made from a fused DNA sequence encoding both afirst polypeptide which catalyzes the reaction of glycine with ATP toform glycyl-adenylate and a second polypeptide which reacts glycyladenylate with tRNA^(GLY) to obtain the glycine-charged tRNA. These twopolypeptides are the alpha and beta subunits of glycine tRNA synthetasewhich has an α₂ β₂ quaternary structure. The two subunits, in the E.coli genome, are encoded by a single dicistronic gene. Toth linked thecoding region of the alpha chain to the coding region of the beta chainby means of a linker encoding six amino acids. See also Toth andSchimmel, J. Biol. Chem., 261: 6643-46 (May 1986).

Ladner, U.S. Pat. No. 4,704,692 describes an expert system for findinglinkers which may be used to convert two naturally aggregated butchemically separated polypeptide chains into a single polypeptide chainwith a similar conformation after folding. This system relies on adatabase containing amino acid sequences for which 3-D structures areknown. The database is examined for candidate amino acid sequences witha span similar in length to the interchain gap to be bridged. Thedirection and orientation of the candidate peptides is then checked. Thealgorithm assumes that these peptides will maintain the same length andorientation regardless of the flanking sequences.

Ladner, WO88/06601 presents a hypothetical approach to the preparationof "pseudodimeric" analogues of dimeric repressor proteins. In essence,an amino acid linker is introduced to convert the dimeric molecule intoa single chain. According to Ladner, this linker may be designeddirectly by the method of the '692 patent; alternatively, thelinker-encoding DNA is a random oligonucleotide and in vivo selection isused to find a pseudodimer whose linker permits the molecule to foldcorrectly and bind sequence-specifically to DNA.

Hallewell, et al., J. Biol. Chem., 264: 5260-68 (1989) prepared ananalogue of CuZn superoxide dismutase. Each dismutase molecule is adimer of two identical subunits; a copper ion and a zinc ion areliganded to the subunit. The dimer interaction in CuZn superoxidedismutase is so strong that the subunits have not been separated withoutinactivating the enzyme. The enzyme has considerable conformationalsimilarity to immunoglobulins; Hallewell, et al., joined two humansuperoxide dismutase genes, either directly or with DNA encoding a19-residue human immunologlobulin IgA1 hinge region and expressed thefused genes in a transformed host. In attempting to express the directlyjoined genes, recombination occurred to eliminate one of the tandemgenes in some plasmid molecules. Hallewell, et al., postulated that thedirect connection distorted the dimer, causing the exposure ofhydrophobic areas which then had a toxic effect. This would haveprovided selection pressure favoring gene deletion. No recombination wasdetected with the IgA1 linker construction.

Unfortunately, it cannot be assumed that a pseudodimeric fusion proteincontaining a peptide linker will fold properly so to be a functionalequivalent of its parental heterodimer.

G. Expression of Soluble Proteins

Efforts to produce heterologous proteins in transformed cells sometimesresult in the precipitation of some or all of the protein as insolubleinclusion bodies, also known as refractile bodies. See, e.g., Paul, etal., Eur. J. Cell Biol., 31:171-174 (1983) (human proinsulin/E. colitrpE fusion protein); Denefle, et al., Gene, 56:61-70 (1987)(angogenin); Langley, et al., Eur. J. Biochem., 163:313-321 (1987)(bovine growth hormone); Petrov, et al., Biology of the Cell, 61:1-4(1987) (calcitonin); Richardson, et al., Biochim. Biophys. Acta,950:385-94 (1988) (ricin B chain); Davis, et al., Biochemistry,26:1322-26 (1987) (tumor necrosis factor); Lee, et al., Biochim.Biophys. Res. Commun., 151:598-607 (1988) (gamma interferon); Meng, etal., J. Chromatogr., 443:183-92 (1988) (Somatomedin C); Tsuji, et al.,Biochemistry, 26:3129-34 (1987) (interleukin-2). The term "refractile"refers to the ability to observe these bodies by phase contrastmicroscopy. Frequently, this insoluble protein retains only a fractionof the expected biological activity, possibly due to incorrect folding.It has been suggested that inclusion bodies are formed when molecules ofpartially folded proteins interact with each other faster than they canfold into their native, active conformation. Kruger, et al., Biopharm,40 (March 1989); Haase-Pettingell and King, J. Biol. Chem., 263:4977-83(1988). "Factors contributing to the formation of inclusion bodies inrecombinant bacteria remain obscure and it is not easy to predict thephysical state of the product of a newly expressed eukaryotic gene in E.coli." Petrov, supra.

While the formation of these inclusion bodies results in enrichment ofthe recombinant protein, and is therefore sometimes desirable, it alsonecessitates solubilization of the aggregates and regeneration of theprotein's biological activity. Petrov, supra at 4, comments, "sometimesthese obstacles seem to be the most critical point of the recombinanttechnology."

Attempts have been made to solubilize and renature these proteins.Wetzel, U.S. Pat. No. 4,599,197; Builder, U.S. Pat. No. 4,620,948;Olson, U.S. Pat. No. 4,511,503; Jones, U.S. Pat. No. 4,512,922. However,such efforts can be laborious and uncertain of success. See Giantini andShatkin, Gene, 56:153-160 (1987). As stated by Weir and Sparks, Biochem.J., 245:85-91 (1987), "proteins vary considerably in their optimalconditions for renaturation; various factors such as pH, saltconcentration and type, rate of removal of denaturant, concentration ofthe target protein and of contaminants may strongly affect the recoveryof authentic protein." These complications are avoided if the protein ofinterest is expressed in soluble form.

Gatenby, et al., Eur. J. Biochem., 168: 227-31 (1987) has discusseddifficulties in the preparation of the higher plant enzymeribulose-bisphosphate carboxylase. This enzyme has the subunit structureL₈ S₈, where L is a large subunit and S is a small subunit. In nature, abinding protein apparently maintains L in soluble form prior to assemblywith S. Attempts to assemble an active higher plant RuBPCase in E. colihave been frustrated by the formation of an insoluble, inactiveaggregate of L.

H. Bacterial Expression of Human Alpha and beta Globins

Nagai and Thorgerson (Nature, 309:810-812, 1984) expressed in E. coli ahybrid protein consisting of the 31 amino-terminal residues of thelambda cII protein, an Ile-Glu-Gly-Arg linker, and the complete humanbeta globin chain. They cleaved the hybrid immediately after the linkerwith blood coagulation factor Xa, thus liberating the beta-globin chain.Later, Nagai, et al., P.N.A.S. (U.S.A.), 82:7252-55 (1985) took therecombinant DNA-derived human beta globin, naturally derived human alphaglobin, and a source of heme and succeeded in producing active humanhemoglobin. Because the alpha globin was derived from erythrocytes, thefinal product may contain undesirable erythrocyte membrane constituents.

More recently, an efficient bacterial expression system for human alphaglobin was reported. GB 8711614, filed May 16, 1987; see copending Ser.No. 07/194,338 and WO 88/09179. This led to the production of whollysynthetic human hemoglobin by separate expression of the insolubleglobin subunits in separate bacterial cell lines, and in situ refoldingof the chains in the presence of oxidized heme cofactor to obtaintetrameric hemoglobin. This procedure is laborious and low in yield. Itrequires the use of denaturing solvents (urea and guanidine), andchemical reduction of ferric ion to the ferrous state (see example). Oneobject of the present invention is to overcome these disadvantages.

While human alpha and beta globins may be expressed separately in E.coli, Walder, Proceedings, Biotech U.S.A. 1988 (San Franciso, Nov.14-16, 1988) warns at page 360, "isolated alpha and beta globin! chainsare unstable and tend to precipitate." If human alpha and beta globinare not produced in soluble form, they must be solubilized withdenaturing agents and then refolded to restore activity. Moreover, whena wild-type alpha globin gene is expressed in E. coli, alpha globinaccumulates only slowly. It is not certain whether this is due toinefficient translation or to the action of host proteases, but WO88/09179 teaches that this problem may be overcome by fusing a shortsection of the beta globin gene to the alpha globin gene, so that ahybrid protein is produced. This hybrid protein must then be cleaved,e.g., with a protease, to release the globin. If the protease is notcompletely selective (perhaps because of contamination by otherproteases), the desired cleavage product may not be the only oneobtained. In any event, that product must be separated from other E.coli polypeptides, and any contaminants associated with the protease.

Sperm whale myoglobin has been expressed in E. coli, demonstrating thatbacteria can incorporate prosthetic heme groups into a protein expressedfrom a cloned eukaryotic gene. Springer and Sligar, PNAS (U.S.A.)84:8961-65 (1987). Walder says, "it remains to be seen if hemoglobin canbe similarly made if both the alpha and beta chains are expressed withinthe same cell." While there is a high degree of tertiary structuresimilarity between myoglobin (a single chain protein) and the individualalpha and beta globin subunits of hemoglobin is a heterotetramericprotein, in which the primary globin sequences have no more than a 27%homology to myoglobin moreover and myoglobin is now known to enjoysignificantly higher stability than either alpha or beta globin. Thus,it could not be predicted that co-expression of alpha- and beta-globinin the same cell would result in intracellular assembly of a functionalhemoglobin, which requires proper folding of the alpha and beta chainsand incorporation of heme.

I. Human Gene Expression in Yeast, Generally

A number of human proteins have been expressed in transformed yeastcells, especially Saccharomyces cerevisiae, either cytoplasmically or bysecretion into the culture medium. King, et al., Biochem. Soc. Transac.,16:1083-1086 (1988). But success is not guaranteed. Thim, et al., FEBSLett., 212:307-312 (1987) experienced difficulty in obtaining properlycrosslinked insulin from yeast cells in which the intactproinsulin-encoding gene had been inserted. They overcame this problemby constructing a modified proinsulin gene which encoded the B and Achains linked by a hexapeptide spacer. The product of this gene wascleaved and the two chains were properly folded and crosslinked by thecells.

Richardson, et al., Biochim. Biophys. Acta, 950:385-94 (1988) expressedthe B chain of the heterodimeric protein ricin in E. coli. They reportedthat it was hard to obtain high levels of secretion of a yeast alphafactor leader/ricin B chain fusion protein. No attempt was made toco-express and assemble the ricin A and B chains.

Murakami, et al., DNA, 6:189-97 (1987) reported production of aheme-containing fused enzyme in transformed yeast cells.

Horwitz, et al., PNAS (U.S.A.), 85:8678-82 (November 1988) described theconstruction of yeast strains which secrete functional mouse variableregion/human IgG1 constant region chimeric antibodies into the culturemedium. They characterize their paper the first report of the secretionof a foreign multimeric or heterodimeric protein in yeast. But see alsoCarlson, Mol. Cell. Biol., 8:2638-46 (June 1988), showing transcriptionand translation of heavy and light-chain cDNAs into polypeptides whichassociate and bind antigen.

Beggs, et al., Nature, 283:835 (1980) attempted to express a chromosomalrabbit beta globin gene in S. cerevisiae. However, these yeast cellswere unable to properly splice the intron-containing globin mRNAtranscript.

No admission is made that any reference cited herein is prior art. Thedescription of the work and the citation of publication date are basedsolely on the published information and the applicants reserve the rightto question the accuracy of that information.

SUMMARY OF THE INVENTION

It is the object of this invention to overcome the aforementioneddeficiencies of the prior art. For example, Applicants have achieved thefirst complete expression and assembly of tetrameric (includingpseudotetrameric) hemoglobin in cells which do not produce hemoglobin innature. Prior work has related to the separate expression of alpha andbeta globin and their extracellular combination with heme to formhemoglobin.

A central feature of the present invention is the intracellular assemblyof alpha- and beta-globin-like polypeptides and intracellularincorporation of heme to form a biologically functional hemoglobin-likeprotein. This intracellular assembly is achieved by expressing thealpha-and beta-globin-like polypeptides in the same cell so that theyfold together and incorporate heme. An important characteristic of thisinvention is a substantial reduction in the formation of insolubleglobin aggregates, in particular of beta globin, as compared to what isobserved when globins are separately expressed in E. coli or S.cerevisiae. Co-expression may be achieved from genes on two separate butcompatible plasmids in the same cell, or from two different operons onthe same plasmid, or from a single polycistronic operon.

In one embodiment, the alpha- and beta-globin-like polypeptides areco-expressed in bacterial cells. The corresponding genes may be includedin the same synthetic operon (i.e., driven by one promoter), or placedin separate operons with separate promoters (which may be the same ordifferent). Preferably, expression of the alpha- and beta-globin isenhanced by placing a "ribosomal loader" sequence as hereafter describedbefore each globin gene. This is particularly advantageous in the caseof alpha globin which is more difficult to produce in quantity.

In another embodiment, the alpha- and beta-globin-like polypeptides areco-expressed in yeast cells. Improvements in both the yield of the alphaglobin and the solubility of beta globin are obtained.

A further aspect of the invention is the production of novelintermediates, di-alpha globin and di-beta globin (and mutants thereof),which can be expressed in a cell (including, but not limited to,bacterial and yeast cells) and assembled with each other or with beta-or alpha-globin-like polypeptides, respectively, into a pseudotetramerichemoglobin-like protein. While intracellular assembly is not strictlyrequired, di-alpha and di-beta globin may be considered speciallyadapted to intracellular assembly of a functional hemoglobin sinceexpression of, e.g., a di-alpha globin is analogous in some respects tointracellular assembly of two alpha globin subunits, differing fromassembly as previously discussed in that the association is accomplishedby expression of a covalent peptide linker rather than by noncovalentinteraction of the subunits. Di-alpha and Di-beta-globin-likepolypeptides may be expressed in, preferably, bacterial cells or inyeast cells.

Moreover, the expression of di-alpha or di-beta (genetically stabilized)hemoglobin-like proteins and the utilization of such as a bloodsubstitute, prolongs the half-life of recombinant hemoglobin by reducingextravasation and glomerular filtration of dissociated subunits in vivocompared to native human hemoglobin. Our studies of hemoglobin isexcreted at levels similar to control levels, while a similarrecombinant hemoglobin excretion in rat urine have demonstrated thatgenetically stabilized recombinant human hemoglobin that was not sogenetically stabilized, undergoes significant dissociation into dimersand is excreted at substantially higher levels. Furthermore, geneticstabilization of hemoglobin results in a two fold or greater increase inhalf-life of hemoglobin in the plasma of rats.

The invention further relates to production of octameric hemoglobins,and of certain higher multimers, by linkage of pseudotetramers invarious configurations.

These facets of the invention are now discussed in greater detail.

Yeast Expression of Hemoglobin-Like Proteins

Applicant have discovered that it is possible to produce humanhemoglobin (or mutants thereof) in yeast, especially Saccharomycescerevisiae. The use of a yeast expression system obviates the need toseparate the hemoglobin from bacterial endotoxins. We have also foundthat alpha and beta globins with the correct N-terminal amino acid maybe obtained without first expressing the globin as a part of selectivelycleavable fusion protein. We believe that this is because the yeastenzyme methionyl aminopeptidase is capable of removing the N-terminalmethionine from Met-alpha-globin and Met-beta-globin to expose thedesired N-terminal amino acid (Valine). Production of altered oxygenaffinity mutants as discussed in WO88/09179 is of special interest. Suchmutants may be produced by site-specific mutagenesis of globin genesfollowed by cloning and expression in yeast.

In a preferred embodiment, expression is controlled by a "gal-gap49"hybrid promoter as hereafter defined.

Co-Expression of Alpha and Beta Globin Genes in Yeast Cells

In a preferred embodiment, the alpha and beta globin genes are bothexpressed within the same yeast cell. Expression of the beta globin genealone results in the production of beta globin as a largely insoluble,difficult-to-extract protein comprising less than 2% of the total cellprotein. Expression of the alpha globin gene alone results in productionof alpha globin at very low levels (under 0.5% of the total cellprotein). In neither case is heme incorporated. When, however, the alphaand beta globin genes are co-expressed, the transformed yeast cells foldthe alpha and beta globin chains together and incorporate heme groups toobtain functional recombinant human hemoglobin in soluble form,accumulating to about 10% of the total cell protein, without any changein the promoters operably linked to the genes.

The alpha and beta globin genes may, in turn, be carried on differentplasmids or on the same plasmid within the host cell.

Polycistronic Co-Expression of the Alpha and Beta Globin Genes inBacterial Cells

Applicants have translationally coupled alpha and beta globin genes to asmall "ribosomal loader" gene encoding a small consumable peptide thatwill lead the ribosome directly into the ATG of the desired alpha andbeta globin message and thus enhance translational efficiency. The havealso placed the alpha and beta globin genes in the same operon so theyare transcribed into a single polycistronic mRNA transcript. The globinsare then translated as separate polypeptide chains which subsequentlyare folded together and joined with intracellular heme by transformedcells to form the hemoglobin tetramer. Applicant's method overcomes theproblem associated with separate purification of precipitated alpha andbeta globins.

The polycistronic expression and assembly of a heterooligomeric humanprotein in soluble, active form in a heterologous host has not beenpreviously reported. It is especially noteworthy that this was amammalian protein expressed in a prokaryotic (bacterial) host. It shouldfurther be considered that this protein incorporates prosthetic groups,which add a further complication to the goal of properpost-translational processing.

In one embodiment, Met-FX-alpha globin and Met-FX-beta globin areco-expressed, where FX denotes a leader peptide which a recognition sitefor Factor Xa cleavage activity. FX-alpha globin and FX-beta globinassemble to form a mutant hemoglobin with reversible oxygen bindingactivity, albeit higher in affinity for oxygen than native hemoglobin.Alternatively, the FX leader, or other fused leader, may be cleaved toobtain a duplicate of native Hgb.

In another embodiment, Met-alpha globin and Met-beta globin areco-expressed. This eliminates the need for a cleavage step.

In a third embodiment, des-val-alpha globin and des-val beta globin areco-expressed. Native alpha and beta globin both begin with valine. Thevaline may, however, be replaced with methionine, which is of similarhydrophobicity.

In further embodiments, one or more codons of the native genes arealtered so that a alpha and/or beta globin-related protein characterizedby one or more amino acid differences (insertions, deletions orsubstitutions) from the native species is produced.

Globin Pseudodimers (especially Di-Alpha and Di-Beta Globins) andGenetically Fused Hemoglobin Pseudotetramers, etc.

A new protein, di-alpha globin, has been prepared, which consists of twoalpha globin amino acid sequences covalently connected by peptide bonds,preferably through an intermediate linker of one or more amino acids.surprisingly, these "genetically fused" alpha globin chains were capableof appropriately folding and combining with beta globin and heme toproduce functional hemoglobin analogue. The term "genetically fused"refers to the method of production. Two copies of the globin gene arefused together, preferably with a spacer DNA encoding the amino acidlinker, so the construct directly encodes the desired di-alpha globin.The term "analogue" is used because in native hemoglobin, the alpha1 andalpha2 subunits are noncovalently bound. The analogous preparation ofdi-beta globin has also been accomplished. Methods for preparation of ananalogous α₁ β₂ (or β₂ α₁) globin pseudodimer have been proposed.

The preparation of "genetically fused" hemoglobins avoids thedisadvantages of chemical crosslinking. The latter is inefficient andoften requires deoxygenation of the hemoglobin solution and the presenceof another molecule (e.g., inositol hexaphosphate or 2,3-DPG) to preventcompeting reactions.

In a preferred embodiment, the di-alpha globin and/or the beta globincontain mutations which reduce the oxygen-binding affinity of thehemoglobin analogue in solution so as to approach the oxygen-bindingcharacteristics of whole blood.

The di-alpha hemoglobin advantageously exhibits a substantially longerhalf-life in the circulatory system than does conventional (des-val)recombinant hemoglobin. Preferably, in humans, the half-life exceeds 9hours at a dose of at least 1 gm/kgm body weight. This would be expectedto correspond to a half-life of about 3 hours in rats given a comparabledose.

Since the fusion prevents dissociation of the hemoglobin into αβ dimers,kidney function is protected.

The di-alpha, di-beta and alphabeta globins can be expressed in cellsconventionally used for expression of recombinant proteins especiallybacteria and yeast.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Flowchart for construction of plasmids for expression ofFX-alpha globin (pDL II-62m), FX-beta globin (pDL II-10a), andFX-hemoglobin (pDL II-66a) are schematized.

FIGS. 2a and 2b: Flowcharts for construction of plasmid pDL III-1a (2a-1and 2a-2) bearing dicistronic Des-Val-Alpha globin gene under control ofTac promoter, and polycistronic di-alpha/beta co-expression plasmid pDLIII-47a (2b-1 to 2b-3).

FIG. 3: Flowchart for construction of plasmid for co-expression ofMet-alpha and Met-beta globins, pDL III-13e (3a and 3b).

FIG. 4: oligonucleotides for construction of synthetic FX-alpha andFX-beta globin genes (4a to 4c). The top strand is shown 5' to 3' andthe bottom strand as 3' to 5'. Areas of overlap between complementarysynthetic oligonucleotides are shown as areas where both strands areshown in the same case letters. The PstI site that joins FX-alpha andFX-beta occurs at the overlap of SJH I-35a and SJH I-36b.

FIG. 5: Synthetic gene for expression of Met-FX-alpha and Met-FX-betaglobin (5a to 5c). Region A contains the alpha globin gene and region Bthe beta globin gene. The location of the Factor X sequence and the twoShine-Delgarno sequences (SD#1 and SD#2) in both regions is indicated.Selected restriction sites are also found. The translated amino acidsequences for the ribosomal loader and Met-FX-alpha/and beta-globin aregiven.

FIG. 6: Elution profile and absorbance spectrum for FX-hemoglobin.

FIG. 7: Oligonucleotides for construction of mutant hemoglobins(differing in amino acid sequence from conventional hemoglobin).

FIG. 8: Oligonucleotides for construction of plasmids which do notencode the Factor Xa substrate recognition site.

FIG. 9: Map of Plasmid pDL III-13e

FIG. 10: Oxygen Binding of Des-Fx Hgb

FIGS. 11a and 11b: Plasmids pDL III-14c (11a) and pDL III-38b (11b).

FIG. 12: Shows the sequence of a preferred synthetic gene for expressionof (des-Val)-alpha-(GlyGly)-alpha and des-Val beta globin (12a to 12c).A shows the region (EcoRI to PstI) containing Shine-Delgarno ribosomalbinding sites (SD#1 and SD#2), the sequence expressing the octapeptide(Met . . . Glu) which serves as a cotranslational coupler, and thesequence encoding the two nearly identical alpha globin-likepolypeptides and the interposed Gly-Gly linker. The first alpha globinsequence begins "Met-Leu", that is, it contains an artifactualmethionine, omits the valine which is the normal first residue of maturealpha globin, and continues with the second residue, leucine. The secondalpha globin sequence begins "Val-Leu", immediately after the underlined"Gly-Gly" linker. Start and stop codons are underlined. B shows theanalogous region (PstI to HindIII) containing the coding sequence fordes-Val beta globin. A and B are connected at the PstI site to form asingle polycistronic operon.

FIG. 13: Shows the structure of the final expression vector pDL III-47a."PTac" is the Tac promoter, and "ampicillin" is the ampicillinresistance gene. FIG. 13a shows an XbaI-BamHI fragment of PDL III-47a.

FIG. 14: Plasmid pSGE0.0E4

FIG. 15: Plasmid pSGE1.1E4

FIG. 16: Plasmid pDL IV-67a

FIG. 17: Plasmid pJR VI-54a

FIG. 18: Plasmid pDL di-alpha/beta/beta

FIG. 19: Flowchart showing the construction of various expressionvectors featuring lambda P_(L) regulation of various polycistronicglobin operons (19a to 19c).

FIG. 20: Nucleotide Sequence of GAL-GAP promoter, with restriction sitesindicated (20a and 20b). The region from SphI to EcoRV contains asynthetic GAL₁₋₁₀ regulatory region (M. Johnston and R. Davis. 1984.Molecular and Cellular Biology, 4:1440-1448). The UAS is in the regionnumbered 29-63 on this Figure. The region from EcoRV to the XbaI sitecontains the consensus GAP491 transcriptional start region, with theapproximate start or transcription being at 395. (L. McAlister and M. J.Holland. 1983. J. Biol. Chem., 260:15019-15027; J. Holland, et al. 1983.J. Biol. Chem., 258:5291-5299.)

FIGS. 21a and 21b: Flowcharts showing construction of beta-globinexpression cassette (21a-1 to 21a-4 and 21b-1 to 21b-3).

FIGS. 22a and 22b: Flowcharts showing construction of beta-globinexpression vector pGS4988 (22a-1 and 22a-2) and alpha-globin expressionvectors pGS4688 and pGS4488 (22b).

FIGS. 23a and 23b: Flowcharts (23a-1 and 23a-2 and 23b-1 to 23b-3)showing construction of an alpha-globin expression cassette and ofvectors pGS289 and pGS389 for co-expression of alpha- and beta globinfrom the same plasmid. Note that alpha and beta globin are expressedfrom separate promoters.

FIG. 24: absorption spectra for yeast-produced recombinant and nativehuman hemoglobin.

FIGS. 25a and 25b: Flowchart (25a-1 to 25a-3) showing construction ofdi-alpha/beta hemoglobin yeast expression vector and map of plasmidpGS3089 (25b).

FIG. 26: Map of plasmid pGS3089RGV desβ.

FIG. 27: is a stylized representation of one form of pseudooctamericHgb, in which the octameric hemoglobin is formed by linking orcrosslinking two molecules of an asymmetric di-alpha Hgb.

FIGS. 28a-e: depicts coiled coil crosslinkers suitable for joining (28a)four or (28b) six Hgb tetramers. FIG. 28(c) is a top view of a 4-helicalbundle, with attachment sites marked.

FIG. 29: maps plasmid pSGE1.1.1E4.

FIG. 30: is a flow chart showing the construction of a di-beta Hgbexpression vector, pSGE1.05E4.

FIG. 31: maps plasmid pSGE1.05E4.

FIG. 32: The symmetry-averaged difference map rHb1.1-HbA superimposed onthe atomic model. The map is contoured at plus (solid contours) andminus (broken contours) about 2 times the root-mean-square value of theelectron density prior to symmetry averaging about the molecular dyad.Filled bonds represent the refined coordinates of the deoxy HbA, Fermi,G., Perutz, M., Shaanan, B., Fourme, R., J. Mol. Biol. 175, 159-174(1984). Open bonds and single broken bonds represent approximatecoordinates of the two symmetry-related configurations of the Hb1.1diα-chain termini: α1-Gly-α2 (open bonds) and α2-Gly-a1 (broken bonds).X-ray data to 2.5 Å resolutions were collected from a single crystal ona FAST/MADNES area detector with a rotating anode source. Absorption,Lorentz and polarization corrections, and batch scale and temperaturefactors (batches of 5 degrees rotatin in angle φ), were applied to theraw X-ray data, which were then merged to yield 18231 uniquereflections, or 96% of the total between 10 and 2.5 Å resolution.Friedel pairs were measured for 76% of unique reflections; the R-factoron intensity between Friedel pairs in the reduced data set was 6.6% onintensity. The reduced data were converted to amplitudes and scaled tonative deoxy HbA data with an R-Factor of 16%. The difference map wascalculated with phases obtained from the atomic model of deoxy Hb.

FIG. 33 In vivo half-life of rHb1.1 and rHb1.0. The fraction of ³H-rHb1.0 or rHb1.1 remaining in the intravascular compartment is shownin the y axis. The value for 100% was determined from samples taken 5min. after administration of the hemoglobin samples. Radioactivehemoglobin was prepared by adding 5 C mCi or ³ H-leucine (Amersham,1100-1500 Ci/mMole) to 2 L fermenters of E. coli JM109 pSGE1.1-E4! orJM109 pSGE1.0-E4! 15 min prior to induction with IPTG. Hemoglobin waspurified essentially as described, Hoffman, S. J., Looker, D. L.,Roehrich, J. M., Cozart, P., Durfee, S., Stetler, G., Proc. Nat. Acad.Sci. (U.S.A.) 87, 8521-25 (1990), and combined with unlabeled rHb1.1 orrHb1.0 to adjust the specific activity to 8.3×10³ dpm/mg (rHb1.1) and9.5×10³ dpm/mg (rHb1.0). Eight Sprague-Dawley rats (200-300 gm) in twogroups of four were administered either 1 ml of ³ H-rHb1.1 (156 mg/ml)through a venous catheter. Blood samples (0.2 ml) were removed at 5min., 30 min., 1,2,3,4,5,6,7, and 8 hours from an arterial catheter. Theanimals were anesthetized with a mixture of ketamine hydrochloride andxylazine by intramuscular injection prior to insertion of catheters andallowed to recover for three days prior to administration of hemoglobinsolutions.

FIG. 34 Map of plasmid pSGE1.0E4

FIG. 35 Schematic showing how cysteine mutations can favor formation ofoctamer without genetic fusion of subunits.

FIG. 36 Proposed alpha₁ -beta₂ globin pseudodimer

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hemoglobin Structure

The structure of conventional hemoglobin is well known. We herewithincorporate by reference the entire text of Bunn and Forget, eds.,Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders Co.,Philadelphia, Pa.: 1986) and of Fermi and Perutz "Hemoglobin andMyoglobin," in Phillips and Richards, Atlas of Molecular Structures inBiology (Clarendon Press: 1981).

The primary structure of a polypeptide is defined by its amino acidsequence and by identification of any modifications of the side chainsof the individual amino acids.

About 92% of the normal adult human hemolysate is Hgb A (designatedalpha2 beta2, because it comprises two alpha and two beta chains). Thealpha chain consists of 141 amino acids (See FIG. 12). The iron atom ofthe heme (ferroprotoporphyrin IX) group is bound covalently to theimidazole of his 87 (the "proximal histidine"). The beta chain is 146residues long (see FIG. 12) and heme is bound to it at his 92.

Other recognized hemoglobin species are Hgb A₂ (α₂ α₂ δ₂), Hgb A_(1a),Hgb A_(1b), and Hgb A_(1c), as well as the rare species Hgb F (α₂gamma₂), Hgb Gower-1 (Zeta₂ epsilon₂), Hgb Gower-2 (alpha₂ epsilon₂),Hgb Portland (Zeta₂ gamma₂), and Hgb H (beta₄) and Hgb Bart (gamma₄).They are distinguished from Hgb A by a different selection ofpolypeptide chains.

Segments of polypeptide chains may be stabilized by folding into one oftwo common conformations. the alpha helix and the beta pleated sheet. Inits native state, about 75% of the hemoglobin molecule is alpha-helical.Alpha-helical segments are separated by segments wherein the chain isless constrained. It is conventional to identify the alpha-helicalsegments of each chain by letters, e.g., the proximal histidine of thealpha chain is F8 (residue 8 of helix F). The non-helical segments areidentified by letter pairs, indicating which helical segments theyconnect. Thus, nonhelical segment BC lies between helix B and helix C.In comparing two variants of a particular hemoglobin chain, it may beenlightening to attempt to align the helical segments when seeking tofind structural homologies. For the amino acid sequence and helicalresidue notation for conventional human hemoglobin A_(o) alpha and betachains, see Bunn and Forget, supra, and Table 1 herein.

The tertiary structure of the hemoglobin molecule refers to the stericrelationships of amino acid residues that are far apart in the linearsequence, while quaternary structure refers to the way in which thesubunits (chains) are packed together. The tertiary and quaternarystructure of the hemoglobin molecule have been discerned by X-raydiffraction analysis of hemoglobin crystals, which allows one tocalculate the three-dimensional positions of the very atoms of themolecule.

In its unoxygenated ("deoxy", or "T" for "tense") form, the subunits ofhemoglobin (alpha1, alpha2, beta1, and beta2) form a tetrahedron havinga twofold axis of symmetry. The axis runs down a water-filled "centralcavity". The subunits interact with one another by means of Van derWaals forces, hydrogen bonds and by ionic interactions (or "saltbridges"). The alpha1beta1 and alpha2beta2 interfaces remain relativelyfixed during oxygenation. In contrast, there is considerable flux at thealpha1beta2 (and alpha2beta1) interface. In its oxygenated ("oxy", or"R" for "relaxed" form), the intersubunit distances are increased.

The tertiary and quaternary structures of native oxyhemoglobin anddeoxyhemoglobin are sufficiently well known that almost all of thenonhydrogen atoms can be positioned with an accuracy of 0.5 Å or better.For human deoxyhemoglobin, see Fermi, et al., J. Mol. Biol., 175: 159(1984), and for human oxyhemoglobin, see Shaanan, J. Mol. Biol., 171: 31(1983), both incorporated by reference.

While analyses of hemoglobin structure tend to focus on the alpha-betainterfaces, it is known that the distance between the amino terminus ofone alpha subunit and the carboxyl terminus of the other is about 5.6 Åin the deoxy configuration and 3.3 Å in the oxy configuration.

Definitions

For the purpose of the appended claims, a hemoglobin-like protein is anoxygen binding protein with a plurality of heme prosthetic groups andcomprising one or more heterotetramers composed of (a) two alphaglobin-like and two beta globin-like polypeptides, (b) one di-alphaglobin-like and two beta globin-like polypeptides, (c) two alphaglobin-like and one di-beta globin-like polypeptides, (d) one di-alphaglobin-like and one di-beta globin-like polypeptides, (e) one fusedalpha/beta globin-like polypeptide and separate alpha and betaglobin-like polypeptides, or (f) two fused alpha/beta globin-likepolypeptides. A polypeptide of one tetramer may be crosslinked orgenetically fused to a polypeptide of another tetramer. A hemoglobin issaid to be multimeric if it comprises more than four globin subunits ordomains. The term "multimeric" thereby includes octameric hemoglobin (2tetramers), as well as higher multimers.

A human alpha globin-like domain or polypeptide is native human alphaglobin or a mutant thereof differing from the native sequence by one ormore substitutions, deletions or insertions, while remainingsubstantially homologous (as hereafter defined) with human alpha globin,and still capable of incorporating heme and associating with betaglobin. A beta globin-like domain or polypeptide is analogously defined.Subunits of animal hemoglobins or mutants thereof which are sufficientlyhomologous with human alpha or beta globin are embraced by the term"human alpha or beta globin-like domain or polypeptide." For example,the subunits of bovine hemoglobin are within the scope of these terms.The alpha- and beta-globin-like polypeptides may be referred tocollectively as "globins". For the sake of convenience the term"polypeptide" may refer to a unitary chain or to a domain of a longerpolypeptide chain.

A "genetically fused hemoglobin" is a hemoglobin-like protein comprisingat least one "genetically fused globin-like polypeptide" (globinpseudooligomer), the latter comprising two or more globin-like domainswhich may be the same or different. A di-alpha globin-like polypeptideis one which consists essentially of two alpha-globin-like polypeptidesequences (domains) connected by peptide bonds between the normalC-terminus of the first alpha-globin-like polypeptide (domain) and thenormal N-terminus of the second alpha-globin-like polypeptide (domain).These two sequences may be directly connected, or connected through apeptide linker of one or more amino acids; the term "peptide bonds" isintended to embrace both possibilities. Apha globin chains crosslinkedat the N- and C-terminals other than by peptide bonds (e.g., by DIDS)are not di-alpha globins. The di-alpha globin-like polypeptide must becapable of folding together with beta globin and incorporating heme toform functional hemoglobin-like protein. The di-beta globin-likepolypeptide is analogously defined. A di-alpha or di-beta globin-likepolypeptide with a mutation in only one of the component domains iscalled "asymmetric".

It is also possible to provide an "alpha/beta-globin-like pseudodimer"in which an alpha globin-like domain is connected by peptide bonds to abeta globin-like domain. This "alpha/beta globin-like polypeptide", andthe di-alpha and di-beta globin-like polypeptides, may collectively bereferred to as "pseudodimeric globin-like polypeptides" or as"diglobins". By extension, a hemoglobin-like protein comprising adi-alpha, a di-beta, or a alpha/beta globin-like polypeptide is a"pseudotetramer".

In determining whether a polypeptide is substantially homologous toalpha (or beta) globin, sequence similarity is an important but notexclusive criterion. Sequence similarity may be determined byconventional algorithms, which typically allow introduction of a smallnumber of gaps in order to achieve the best fit. Preferably, thealpha-globin-like polypeptides or domains) of the present invention haveat least about 75% sequence identity with wild-type human alpha globin.However, a polypeptide of lesser sequence identity may still beconsidered "substantially homologous" with alpha globin if it has agreater sequence identity than would be expected from chance and alsohas the characteristic higher structure of alpha globin and similarbiological activity. (Note that, as elsewhere explained, an alterationin oxygen affinity (P50), intravascular retention, or cooperativity maybe desired, and does not render the mutant nonhomologous if it can stillcontribute to reversible oxygen-binding activity.) By way of comparison,Artemia's heme-binding domains are considered homologous with myoglobineven though the primary sequence similarity is no more than 27%, asalignment of the heme-binding domains around their conserved residuesand the residues conserved in other hemoglobins (i.e., involved in hemecontacts or in determining the relationship of the helical segments toeach other) suggested that the Artemia domains possessed the classicalglobin helices A to H with their corresponding turns, as well as variousconserved globin family residues. Also, among the serine proteaseinhibitors, there are families of proteins recognized to be homologousin which there are pairs of members with as little as 30% sequencehomology.

Over a hundred mutants of human hemoglobin are known, affecting both thealpha and beta chains, and the effect of many of these mutations onoxygen-binding and other characteristics of hemoglobin are known. Thehuman alpha and beta globins themselves differ at 84 positions. Inaddition, interspecies variations in globin sequence have beenextensively studied. Dickerson, Hemoglobin: Structure, Function,Evolution and Pathology, ch. 3 (1983) reported that in 1982, the 60known vertebrate alpha globins had identical residues at 23 of their 141positions, while for the 66 vertebrate beta globins considered, 20 ofthe 146 amino acids are identical. The 60 vertebrate myoglobins, whichalso belong to the globin family, had 27 invariant amino acids out of153 positions. If only mammals are considered, then the invariant aminoacids are 50/141 for the alpha globins, 51/146 for the beta globins, and71/153 for the myoglobins. Invariant positions cluster around thecenters of activity of the molecule: the heme crevice and theintersubunit contacts. Of the variable amino acids, some diverge fromthe consensus sequence for only a small fraction of the speciesconsidered.

The number of total differences between human alpha globin and selectedother vertebrate alpha globins is as follows: rhesus monkey (4), cow(17), platypus (39), chicken (35), human zeta (embryonic) (61), carp(71), and shark (88). For invertebrate globins the divergences are sealamprey (113), mollusk (124), Glycera (marine bloodworm) (124) andChironomus (midge) (131). Turning to the beta globin family, thedifferences of human beta globin from other vertebrate beta globins arerhesus monkey (8), human delta globin (10), cow beta globin (25), cowgamma globin (33), human gamma globin (39), human epsilon (embryonic)globin (36), platypus (34), chicken (45), shark (96), sea lamprey (123),mollusc (127), Glycera (125) and Chironomus (128).

Many of these differences may be misleading--variable amino acids mayexhibit only "conservative substitutions" of one amino acid for another,functionally equivalent one. A "conservative substitution" is asubstitution which does not abolish the ability of a globin-likepolypeptide (or domain) to incorporate heme and to associate with alphaand beta globin subunits to form a tetrameric (or pseudotetrameric)hemoglobin-like protein which, in keeping with the definition thereof,will reversibly bind oxygen. The following resources may be used toidentify conservative substitutions (and deletions or insertions):

(a) data on functional hemoglobin mutants (over a hundred such mutantsexist);

(b) data on sequence variations among vertebrate, especially mammalian,alpha globins and beta globins;

(c) data on sequence variations among vertebrate, especially mammalian,myoglobins;

(d) data on sequence variations between vertebrate and invertebrateglobins, or among the invertebrate globins;

(e) data on the three-dimensional structures of human hemoglobin andother oxygen-binding proteins, and molecular modelling software forpredicting the effect of sequence changes on such structures; and

(f) data on the frequencies of amino acid changes between members offamilies of homologous proteins (not limited to the globin family). See,e.g., Table 1-2 of Schulz and Schirmer, Principles of Protein Structure(Springer-Verlag: 1979) and FIGS. 3-9 of Creighton, Proteins: Structureand Molecular Properties (W. H. Freeman: 1983).

While the data from (a)-(α) is most useful in determining tolerablemutations at the site of variation in the cognate proteins, it may alsobe helpful in identifying tolerable mutations at analogous siteselsewhere in the molecule. Based on the data in category (f), thefollowing exchange groups may be identified, within which substitutionsof amino acids are frequently conservative:

I small aliphatic, nonpolar or slightly polar residues-Ala, Ser, Thr(Pro, Gly)

II negatively charged residues and their amides-Asn Asp Glu Gln

III positively charged residues-His Arg Lys

IV large aliphatic nonpolar residues-Met Leu Ile Val (Cys)

V large aromatic residues-Phe Tyr Trp

Three residues are parenthesized because of their special roles inprotein architecture. Gly is the only residue without a side chain andtherefore imparts flexibility to the chain. Pro has an unusual geometrywhich tightly constrains the chain. Cys can participate in disulfidebonds which hold proteins into a particular folding. Note that Schulzand Schimer would merge I and II above. Note also that Tyr, because ofits hydrogen bonding potential, has some kinship with Ser, Thr, etc.

In general, functionality is less likely to be affected by mutations atsurface residues, at least those not involved in either the heme creviceor the subunit contacts. In addition, "loops" connecting alpha helices,as well as free amino or carboxy termini, are more tolerant of deletionsand insertions.

A "Met FX alpha globin" is an alpha globin-like polypeptide comprisingan N-terminal methionine, a oligopeptide which acts as a recognitionsite for Faster Xa (e.g., Ile-Glu-Gly-Arg), and an alpha globin-likesequence (e.g., Val-His-Leu-Thr-Pro . . . ) which may correspond towild-type alpha globin or to a mutant thereof as taught herein. The term"Met FX alpha globin" is sometimes abbreviated as "FX alpha globin". "FXbeta globin" is an analogously defined beta globin-like polypeptide.

"Met-alpha globin" is an alpha globin-like polypeptide with an extraN-terminal methionine. The second amino acid is valine, which is thefirst amino acid of mature wild-type alpha globin. Met-beta globin isanalogously defined. A "Des-FX alpha globin" gene (or "dFX alphaglobin") is a Met-alpha globin gene obtained by excising the FX codonsfrom a Met-FX alpha globin gene. Note that "Met-Hgb" is used to refer tomethionyl. Hgb formed from methionyl-alpha globin and methionyl-betaglobin.

"Des-Val-alpha globin" (or "dVal alpha globin") is an alpha globin-likepolypeptide wherein methionine is substituted for the valine whichbegins the sequence of mature wild-type alpha globin. Des-Val-betaglobin is analogously defined. Des-Val-alpha/alpha globin(di-Des-Val-alpha globin) is a "di-alpha globin" in which a"Des-Val-alpha" sequence is linked via an appropriate peptidyl linker toan alpha globin-like sequence which begins with Val.

The alpha and beta globin-like chains need not correspond exactly insequence to the alpha and beta globins of "conventional" hemoglobin.Rather, mutations may be introduced to alter the oxygen affinity orstability of the hemoglobin, or the ease of expression and assembly ofthe individual chains. By way of example and not limitation, severalmutant hemoglobins have been prepared by the method of this invention.Guidance as to further mutations is provided, e.g., by Hoffman andNagai, U.S. Pat. No. 5,028,588, and Ser. No. 07/443,950, incorporated byreference herein.

The term "low affinity hemoglobin-like protein" refers to ahemoglobin-like protein having a P₅₀ which is at least 10% greater thanthe P₅₀ of cell free hemoglobin A₀ under the same conditions.Preferably, the protein, if used as a blood substitute, qualifies as alow affinity protein, and more preferably, its P₅₀ is closer to the P₅₀of whole blood cells than to that of cell free hemoglobin.

The term "high affinity hemoglobin-like protein" refers to ahemoglobin-like protein having a P₅₀ which is at least 10% less than theP₅₀ of cell free hemoglobin A₀ under the same conditions.

Low and High Affinity Mutants

Low affinity mutant hemoglobins, i.e., those with "right shifted" oxygenequilibrium binding curves relative to cell-free conventionalhemoglobin, have many potential uses. Most notably, mutant hemoglobinsthat have an oxygen affinity similar to whole red blood cells may beused as an oxygen-carrying transfusion substitute in place of donatedred blood cells, eliminating the risk of infection and alleviatingproblems with supply. Cell-free native human hemoglobin cannot functionas a transfusion substitute, among other reasons because oxygen is boundtoo tightly. In addition, because cell-free hemoglobin solutions do notneed to be cross-matched and are expected to have a longer shelf lifethan whole blood, low affinity hemoglobin solutions may be widely usedin situations where whole blood transfusion is not feasible, for examplein an ambulance or on a battlefield. Mutant hemoglobins that have aneven lower oxygen affinity than red blood cells may in fact deliveryoxygen more effectively in many situations. Mutant hemoglobins that havea somewhat higher oxygen affinity than whole blood (but a lower affinitythan cell-free native human hemoglobin) will still function as anadequate transfusion substitute and may in fact deliver oxygen moreeffectively than red blood cells in some situations. This is becauseoxygen is released directly to plasma form hemoglobin-based solutions,without the need to diffuse through the red cell membrane, and becausecell-free hemoglobin may penetrate into regions not accessible to redblood cells. As an example, low affinity mutant hemoglobin is expectedto deliver oxygen effectively during coronary artery balloon angioplastyprocedures, whereas circulation of red blood cells is obstructed duringsuch procedures. Low affinity mutant hemoglobin may also be useful as aperfusion component in organ preservation prior to transplantation or asa mammalian cell culture additive.

High affinity mutant hemoglobin may have utility in other situations.For example, perfluorocarbon-based blood substitute preparations areunder clinical study for enhancement of radiation therapy and certainchemotherapy treatments of solid tumors (Dowling, S., Fischer, J. J.,and Rockwell, S. (1991) Biomat. Art. Cells Immobil. Biotech, 19, 277;Herman, T. S. and Teicher, B. A. (1991) Biomat. Art. Cells and Immobil.Biotech, 19, 395; Holden, S. A., Teicher, B. A. and Herman, T. S. (1991)Biomat. Art. Cells and Immobil. Biotech, 19, 399.) The basis of theseinvestigations is the fact that oxygen is a required component of thecell toxicity action of radiation and certain chemotherapy reagents.Solid tumors frequently exhibit extremely low partial oxygen pressure inthe interior of the tumor, rendering therapy inefficient.Perfluorocarbon-based oxygen-carrying solutions appear to dramaticallyenhance certain tumor therapies, and hemoglobin-based blood substitutesare expected to have a similar utility. It is likely that cell-freehemoglobin unlike whole red blood cells, will be able to penetrate theinterior region of tumors for delivery of oxygen. Actual percent ofoxygen released by a cell-free hemoglobin preparation is not a directfunction of P₅₀ but rather depends on the shape of the oxygenequilibrium binding curve between the two pressures representing thepartial oxygen pressure of the lungs (where oxygen is loaded ontohemoglobin) and the partial pressure of the tissue where oxygen isunloaded. Therefore, it is possible that a high affinity mutanthemoglobin would be preferred as a tumor therapy adjuvant. A highaffinity hemoglobin would retain its bound oxygen throughout the normalcirculatory system, where partial oxygen pressure remains relativelyhigh, but release its oxygen in the extremely oxygen-depleted tumorinterior. Normal or low affinity hemoglobin might have less hemoglobinavailable for release by the time it reaches the interior of the tumor.

Possible low affinity mutants are discussed in detail, by way of exampleand not of limitation, in Table 1 (natural low affinity hemoglobinmutants) and Table 2 (candidate non-naturally occurring low affinityhemoglobin mutants) of Hoffman, et al., U.S. Pat. No. 5,028,588. Lowaffinity mutants of particular interest are the Presbyterian (betaLys¹⁰⁸) beta Phe⁶³, beta Ile⁶⁷, and Kansas (beta Thr¹⁰²) mutants.

Naturally occurring high affinity hemoglobin mutants are also known, seeBunn and Forget, Table 14-1, and candidate non-naturally occurring highaffinity hemoglobin mutants may be proposed in view of the known mutantsand hemoglobin structure. For example, a naturally occurring highaffinity hemoglobin mutant is hemoglobin Providence having a Lys82Asnsubstitution in the human beta globin protein. Particularly preferredhigh affinity mutants are set forth in Table 400.

It should be noted that genetic fusion and crosslinking can affectoxygen binding affinity.

Cysteine Mutations and Disulfide Bridge Formation

Cysteine mutations are of value for increasing the stability of thetetramer (See U.S. Pat. No. 5,028,588 and Ser. No. 07/443,950. They alsofacilitate constructing poly(tetrameric) (n>=2) hemoglobins withincreased intravascular half-life. This is because the cysteines onadjacent tetramers (including pseudotetramers) can be oxidized to form adisulfide bridge, covalently coupling the tetramers.

A variety of sites are available for introduction of cysteines into ahemoglobin-like protein.

The criteria governing site selection are: (1) the mutation does notaffect functionality; (2) the side chain is accessible to water in oxyor deoxy structure; (3) the site should lie on the surface of the foldedprotein; (4) the sulfhydryl of the side chain should extend away fromthe surface rather than toward the interior of the molecule; (5) thesite should be in a portion of the molecule that is not directlyinvovled in the R→T transition; (6) the change should be in a portion ofthe molecule that does not have a tightly fixed position (such regionsgenerally give indistinct X-ray diffraction patterns); (7) the mutationswill not destroy the local secondary structure, i.e., avoid pro→cysmutations, which might result in a refolding problem; and (8) ifpossible, a conservative change should be made such as ser→cys orala>cys. A mutation does not necessarily have to meet all of the aboverequirements to be useful. For example, one might envision a site thatis involved in the R→T transition (cf. 5 above) but confers a beneficialchange in P₅₀ (cf. 1 above) because of that involvement. The mostimportant considerations are that the mutation does not abolish O₂binding and that the cysteine is accessible for participation indisulfide bonding.

Candidate sites on the alpha surface include: his72, asn 78, asn68,ala71, thr67, lys7, lys11, thr8, ala12, thr118, lys16, ala45, glu116,gly15, his112, thr24, glu23, lys60, lys56, his50, gly51, glu53, ser49,asp47, gln54, his45, lys90, ala82, lys61, ala19, his20, asp85, ser81,asp75, asp74, lys139, asp64, and gly18 (total 40 amino acids).

Candidate sites on the beta surfaces includes: asp79, his2, leu3, thr4,glu6, ser9, thr12, ala13, gly16, lys17, val18, asn19, val20, asp21,glu22, lys65, ser72, ala76, his77, asp79, asn80, gly83, ala86, thr87,glu90, lys95, lys59, glu43, ser44, asp47, ser49, thr50, ala53, asp52,lys61, glu121, lys120, thr123, lys66, asp73, ala62, his116, his117,(total 45 amino acids).

There are a number of naturally occurring mutants which already showmutations at these sites. These are listed below:

    ______________________________________                                        Residues   Region          Mutation                                           ______________________________________                                        19         AB1             ALA->GLU                                                                      ALA->ASP                                           54         E3              GLN->ARG                                                                      GLN->GLU                                           71         E20             ALA->GLU                                           75         EF4             ASP->GLY                                                                      ASP->HIS                                                                      ASP->TYR                                                                      ASP->ASN                                           81         F2              SER->CYS                                           47         CE5             ASP->GLY                                                                      ASP->HIS                                                                      ASP->ASN                                           ______________________________________                                    

If the pseudo-octamer (n=2) is formed by directly linking twopseudo-tetramers via a disulfide bond, the halflife in serum may beinfluenced by the rate at which endogenous serum small molecule thiols(such as glutathione) reduce the disulfide bond. The mechanism of thesereactions involves the thiolate anion as the actual reducing species(Creighton, T. E. (1978) Prog. Biophys. Molec. Biol., 33:259-260;Creighton, T. E. (1975) J. Mol. Biol., 96:767; Creighton, T. E. (1977)J. Mol. Biol., 113:313). Thus the rate of reduction will be a functionof the molecular electrostatic environment in the vicinity of thedisulfide bond. A slower rate of reduction would be predicted if thedisulfide was located in an electrostatically negative environment, dueto the repulsion of the thiolate anion. In the case of glutathiene, eventhe unreactive transient protonated species has a net negative chargeand would be repulsed, thus further reducing the rate of disulfidereduction.

A surface or near-surface amino acid residue of di-alpha or di-betahemoglobin that is located in close proximity to a negatively chargedsurface residue might therefore be a good choice for location of asingle cysteine mutation in the di-alpha or di-beta polypeptide.Although formation of the initial disulfide bond between two suchcysteines might also be slower because of repulsion between the negativecharges on the two hemoglobin molecules in the vicinity of thecysteines, the reaction could be facilitated by use of high salt or highpH during the in vitro bond formation reaction. If carried out underdeoxy conditions in a redox buffer, the reaction might also befacilitated by temperature elevation.

Preferred sites for cys mutations proximal to negative charged residues

    ______________________________________                                        alpha ser49                                                                              near asp47 naturally occurring ser49 to arg                                              has normal O.sub.2 affinity                             alpha his20                                                                              near glu23 naturally occurring his20 to tyr,                                             gln, arg have no known                                                        undesirable properties                                  alpha lys16                                                                              near glu116                                                                              naturally occurring lys to glu has                                            normal O.sub.2 affinity                                 alpha his50                                                                              near glu30 naturally occurring his50 to asp                                              has no known undesirable                                                      properties                                              beta thr50 near asp52 naturally occurring thr50 to lys                                              has no known undesirable                                                      properties                                              beta lys65 near asp21                                                         beta asn19 near asp21                                                         ______________________________________                                    

Surface or near-surface cysteine mutations in general are not expectedto have major effects on the functionality of the hemoglobinpseudotetramer. Cysteine mutations would not be expected tosignificantly destabilize alpha helices, and surface residues are notdirectly involved in the oxygen binding properties of hemoglobin. Mostsurface residues undergo considerable motion and are not tightlyconstrained. It should also be noted that because of protein breathingmotions, the cysteine side chain would not necessarily have to pointdirectly into solution to be accessible for disulfide bond formation.

In addition to the use in construction of a pseudo-octamer, there may beadditional uses of surface cysteine mutations. These include: (1)construction of multimeric hemoglobins (n>2) by use of syntheticsulfhydryl reactive peptides with more than two reactive sites; (2)surface cysteine residues could be used to attach chelates that bindradioisotopes for imaging; and (3) surface cysteines could be used toattach bio-active peptides or other therapeutic agents to increase theircirculating half-life, or target their delivery. If the attachment ofthe drug were via a disulfide, the rate of release of the peptide fromits carrier could be controlled by neighboring residues. For uses (2)and (3), restriction to one cysteine per di-alpha or di-beta isunnecessary.

Gene Construction and Expression

The DNA sequences encoding the individual alpha (or di-alpha) and beta(or di-beta) globin chains may be of genomic, cDNA and synthetic origin,or a combination thereof. Since the genomic globin genes containsintrons, genomic DNA must either be expressed in a host which canproperly splice the premessenger RNA or modified by excising theintrons. Use of an at least partially synthetic gene is preferable forseveral reasons. First, the codons encoding the desired amino acids maybe selected with a view to providing unique or nearly unique restrictionsites at convenient points in the sequence, thus facilitating rapidalteration of the sequence by cassette mutagenesis. Second, the codonselection may be made to optimize expression in a selected host. Forcodon preferences in E. coli, see Konigsberg, et al., PNAS, 80:687-91(1983). For codon preferences in yeast, see the next section. Finally,secondary structures formed by the messenger RNA transcript mayinterfere with transcription or translation. If so, these secondarystructures may be eliminated by altering the codon selections.

Of course, if a linker is used to genetically crosslink subunits, thelinker will normally be encoded by a synthetic DNA. While the di-alphaglobin and the beta globin may be expressed separately and then combinedwith each other and heme in vitro, they are preferably placed on oneplasmid.

The present invention is not limited to the use of any particular hostcell, vector, or promoter. The host cell may be a microbial, plant oranimal cell, and, if a plant or animal, it may be in cell, tissue ororgan culture, or a cell of a transgenic or chimeric plant or animal.However, the preferred host cells are bacterial (especially, E. coli)and yeast (especially S. cerevisiae) cells. The promoter selected mustbe functional in the desired host cells. It preferably is an induciblepromoter which, upon induction, provides a high rate of transcription. Apreferred bacterial promoter is the Tac promoter, a trp/lac hybriddescribed fully in DeBoer, U.S. Pat. No. 4,551,433 and commerciallyavailable from Pharmacia-LKB. Other promoters which might be usedinclude the temperature sensitive lambda P_(L) and P_(R) promoters, aswell as the lac, trp, trc, pIN (lipoprotein promoter and lac operatorhybrid), gal and heat shock promoters. The promoter used need not beidentical to any naturally-occurring promoter. Guidance for the designof promoters is provided by studies of promoter structure such as thatof Harley and Reynolds, Nucleic Acids Res., 15:2343-61 (1987) and paperscited therein. The location of the promoter relative to the firststructural gene may be optimized. See Roberts, et al., PNAS (U.S.A.),76:760-4 (1979). The use of a single promoter is favored. Suitable yeastexpression systems are described in detail elsewhere in thisspecification.

The vector used must be one having an origin of replication which isfunctional in the host cell. It desirably also has unique restrictionsites for insertion of the globin genes and the desired regulatoryelements and a conventional selectable marker. A vector may be modifiedto introduce or eliminate restriction sites to make it more suitable forfuther manipulations.

The alpha and beta globin chains may be expressed either directly or aspart of fusion proteins. When expressed as fusion proteins, the lattermay include a site at which they may be cleaved to release the alpha andbeta globin free of extraneous polypeptide. If so, a site sensitive tothe enzyme Factor Xa may be provided, as taught in Nagai and Thorgenson,EP Appl 161,937, incorporated by references herein. Alternatively, thealpha and beta fusion proteins may be synthesized, folded and hemeincorporated to yield a hemoglobin analogue.

The direct expression of the alpha and beta globin subunits isdesirable. Factor Xa is a blood derivative. Preparations of Factor Xamay therefore contain undesirable blood-associated substances oretiologic agents. In any event, the hemoglobin must be separated fromthe Factor Xa.

Nagai and Thorgerson, EP Appl 161,937, incorporated by reference herein,teach the construction of fused genes in which DNA coding for apolypeptide of interest is immediately preceded by DNA encoding acleavage site for Factor Xa, a serine protease. Certain of the peptidesequences to be cleaved by Factor Xa are quoted below (wherein thecleavage site is denoted by an "="):

Ile-Glu-Gly-Arg=Val-His-Leu-Thr CII FxB-globin

Ile-Glu-Gly-Arg=Thr-Ala-Thr-Ser Hu prothrombin

Ile-Glu-Gly-Arg=Thr-Ser-Glu-Asp Bo prothrombin

Ile-Asp-Gly-Arg=Ile-Val-Glu-Gly Hu prothrombin

Ile-Glu-Gly-Arg=Ile-Val-Glu-Gly Bo prothrombin

Ala-Glu-Gly-Arg=Asp-Asp-Leu-Tyr Hu antithrombin III

In the above list, "CIIFXβ-globin" refers to a hybrid fusion proteincomprises the 31 amino-terminal residues of the lambdaCII protein, theFactor Xa recognition sequence "Ile-Glu-Gly-Arg," and the complete aminoacid sequence of human beta globin (which begins "Val-His-Leu-Thr- . . ."). It will be evident from study of FIG. 4 of the present inventionthat FX-alpha and FX-beta globins of Example 1 correspond to the nativeglobin preceded by "Met-Ile-Glu-Gly-Arg."

In bacterial mRNA, the site at which the ribosome binds to the messengeris a polypurine stretch which lies 4-7 bases upstream of the start (AUG)codon. The consensus sequence of this stretch is 5' . . . AGGAGG . . .3', and is frequently referred to as the Shine-Dalgarno sequence. Shineand Dalgarno, Nature, 254: 34 (1975). The exact distance between the SDsequence and the translational start codon, and the base sequence ofthis "spacer" region, affect the efficiency of translation and may beoptimized empirically. Shepard, et al., DNA 1: 125 (1985); DeBoer, etal., DNA 2: 231 (1983); Hui, et al., EMBO J., 3: 623 (1984).

In addition, the SD sequence may itself be modified to alter expression.Hui and DeBoer, PNAS (U.S.A.), 84:4762-66 (1987). Comparative studies ofribosomal binding sites, such as the study of Scherer, et al., NucleicAcids Res., 8:3895-3907 (1987), may provide guidance as to suitable basechanges. If the hemoglobin is to be expressed in a host other than E.coli, a ribosomal-binding site preferred by that host should beprovided. Zaghbil and Doi, J. Bacteriol., 168:1033-35 (1986).

Any host may be used which recognizes the selected promoter andribosomal binding site and which has the capability of synthesizing andincorporating heme. Bacterial and yeast hosts are preferred.

The intracellularly assembled hemoglobin may be recovered from theproducing cells and purified by any art-recognized technique.

Polycistronic Co-Expression of Alpha and Beta Globins and Their AssemblyInto Hemoglobin

In one embodiment, expression of the alpha and beta globin genes isdriven by a single promoter, and the genes are arranged so that apolycistronic messenger RNA transcript is transcribed, from which theseparate alpha and beta globin polypeptides are subsequently translated.However, the present invention includes the co-expression of the alphaand beta globin genes from separate promoters, i.e., the hosttranscribes separate alpha and beta globin mRNAs.

The use of a single promoter is favored on theoretical grounds. Ideally,alpha and beta globin are expressed in stoichiometrically equal amounts.While use of a single promoter does not guarantee equality, iteliminates one unbalancing influence--differences in transcription owingto differences in promoter strength and accessibility. If differences inpromoter strength were minimized by use of two identical promoters onthe same plasmid, plasmid stability would be reduced as there would be apropensity toward recombination of the homologous regions. We note,however, that in preliminary experiments we have co-expressed alpha andbeta globins from separate promoters.

Another justification for using a single promoter is to minimize thenumber of repressor binding sites.

Preferably, the alpha and beta globin genes are arranged so that theribosome will translate the alpha globin cistron first. The rationale isthat there is some basis for believing that alpha globin affects thefolding of beta globin. Nonetheless, the position of the genes may beswitched so that beta globin is synthesized first, as is shown inExample 6.

The stability of the polycistronic mRNA transcript, the efficacy of itstranslation into alpha and beta globin, and the folding of the globinchains into tetrameric hemoglobin may be modified by varying the lengthand base sequence of the intercistronic regions (the region lyingbetween the stop codon of one cistron and the start codon of the nextcistron), the phasing of a second cistron relative to a first cistron,and the position and sequence of the ribosomal binding site for the onecistron relative to the preceding cistron.

In a preferred embodiment, the alpha and beta globin genes are eachpreceded by a short "introductory" cistron or "ribosomal loader" whichfacilities the subsequent translation of the globin cistron. In FIG. 4,region A contains two cistrons and a Shine-Delgarno sequence preceedingeach cistron. The first Shine-Delgarno sequence (SD#1) is bound by theribosome, which then translates the first cistron, a short cistronencoding an octapeptide. (This cistron is referred to as an"introductory cistron or ribosomal loader.) The second cistron is aglobin gene, in this case, an FX alpha-globin gene. The Shine-Delgarnosequence (SD#2) for facilitating translation of the second cistronactually lies within the first cistron. For this reason, the two aresaid to be "translationally coupled". Region B is identical instructure, except that the second cistron encodes FX-beta globin.Between regions A and B is a 43-base intercistronic region. Theintroductory cistrons of regions A and B correspond to the first cistronof the two-cistron expression system denoted pCZ144 in Schoner, et al.,Meth. Enzymol., 153: 401-16 (1987). The present invention is not,however, limited to the particular "starter" cistron taught by Schoner,et al.; other introductory cistrons that allow for restart of high leveltranslation of a following cistron may be used.

Guidance as to the design of intercistronic sequences and as to thelocation of SD sequences may be obtained by comparing the translationalefficiency of spontaneous or controlled mutants of the samepolycistronic operon, as exemplified by Schoner, et al., PNAS, 83:8506-10 (1980). It is also possible to look for consensus features inthe intercistronic regions of different operons. McCarthy, et al., EMBOJ., 4: 519-26 (1985) have identified a translation-enhancingintercistronic sequence in the E. coli atp operon.

The present invention is intended to reduce or avoid the localization ofthe hemoglobin or its component polypeptides into inclusion bodies.Consequently, a further feature of the invention is that the functionalhemoglobin is substantially found (preferably over 80%) in the solublefraction of the cell. It appears that with this invention, over 90% ofthe functional hemoglobin can be so directed when alpha₂ beta₂hemoglobin is assembled from alpha- and beta-globin chains co-expressedfrom a tetracistronic operon as described herein. With di-alpha, beta2hemoglobin, nearly 100% is soluble when expression is induced at 25° C.and less at higher induction temperatures. These percentages reflect thepercent of all di-alpha and beta chains found in the soluble fraction ofthe cell and not actual recovery of protein from the cell.

Expression in Yeast

In another embodiment the present invention relates to the production ofhemoglobin-like molecules in yeast. Our preferred host for expression ofrecombinant human hemoglobin in yeast is Saccharomyces cerevisiae.However, other fungi or yeast may be used for the purpose, such asstrains of Aspergillus or Pichia. For yeast to be a suitable host itmust be capable of being transformed with recombinant vectors, eitherreplicating or integrating types. This allows the insertion of thedesired DNA sequence for the gene of interest. It must also be capableof high density cell growth, in appropriate volume to provide sufficientcell mass to isolate the desired gene product from the desired reactionvessels, where ideally the growth would be easily controlled by severalparameters including nutrient formulation, agitation and oxygen transferand temperature. It is also desirable to be able to induce theexpression of protein synthesis with the manipulation of the media,temperature, or by the addition or consumption of certain chemicals.Finally, to be a suitable host, the yeast must be capable of producingrecombinant proteins, preferably in excess of 1% of the total cellprotein. This allows more facile isolation of the desired recombinantprotein.

With reference to S. cerevisiae, haploid strains of potential useinclude:

    ______________________________________                                        BJY3501    Matα pep4::HIS3 prb1-D 1.6R his3 200 ura3-52                            can1 GAL,                                                          GSY112     As above but Leu2::HISG                                            GSY112 cir As above but cured of 2μ plasmid                                BJY3505    Mata pep4::HIS3 prb1-D 1.6R HIS3 lys2-208                                     trpl-101 ura3-52 gal2 can1                                         GSY113     As above but leu2::HISG                                            R5Y330     Matα pep4-3 prb1-1122 hist7 ura3-52 trp1-289                            can1 gal1                                                          BJY2168    Mata prc1-407 prb1-1122 pep4-3 leu2 trp ura3-                                 52                                                                 BJY1991    Matα prb1-1122 pep4-3 leu2 trp1 ura3-52                      RSY334     Matα reg1-501 pep4-3 prb1-1122 ura3-52 leu2-3,                          112 gal1                                                           RSY214     Matα pep4-3 prb1 ura3-52                                     ______________________________________                                    

To date, strains such as GSY112, GSY113 and RSY334 have been the besthemoglobin producers. Strains such as RSY334 that carry the reg1-501mutation may be particularly important as they uncouple glucoserepression from galactose induction, allowing one to induce with lowerlevels of galactose in the presence of glucose. Because this straincarries the gall mutation it cannot metabolize galactose, so thegalactose concentration remains constant and continues to function as ainducer.

Diploid strains formed from crosses of any of the above compatiblestrains or similar compatible strains may also be useful as they tend tohave faster growth rates than haploid strains.

For example, the following diploid strains, which co-express alpha (ordi-alpha) and beta globins, are described in the Examples:

BJY3505 pGS4988!×RSY330 pGS4688!

BJY3505 pGS4988!×BJY 1991 pGS4688!

Other matings may likewise be used in practicing the present invention.

The use of protease-deficient strains may also be advantageous.

Yeast expression systems can be divided into two main categories: (1)Systems designed to secrete protein and (2) system designed for thecytoplasmic expression of proteins. The advantages of secretion systemsare:

(1) The protein is often easier to purify from culture medium than fromtotal cell extracts.

(2) If the protein has essential disulfide bonds, they are more likelyto form if the protein passes through the secretory pathway. This isthought to be partly due to the presence of protein disulfide isomerasein the endoplasmic reticulum and to a less reducing environment than inthe cytoplasm.

(3) Secretion can also be advantageous if the first amino acid (afterthe initiating methionine) creates a dipeptide sequence that is poorlyprocessed by methionyl aminopeptidase. The addition of a secretorysignal sequence may allow processing by signal peptidase duringsecretion resulting in a protein with the authentic amino acid at theamino terminus of the protein.

The disadvantages of a secretion system are:

(1) Generally, the expression level are much lower than those that canbe obtained by cytoplasmic expression. This seems to be, in part, due toa rate limiting step in secretion.

(2) Not all proteins are secretable.

(3) Often, particularly with S. cerevisiae, misprocessed forms of theprotein accumulate and these can be difficult to purify away fromcorrectly processed forms.

The secretory expression system must commonly used in recombinant yeastis the yeast α-factor secretory signal sequence and the α-factorpromoter. See A. J. Brake. Yeast Genetic Engineering Eds. P. Barr, A.Brake, and P. Valenzuela. Butterworth Publishing, Boston 1989, for areview. The invertase signal sequence coupled to a variety of promotershas also been used to express heterologous proteins in yeast. See G.Stetler et al., Biotechnology, 7:55-60 (1989), R. Smith et al., Science,229:1219-1229 (1985).

The advantages of cytoplasmic expression are:

(1) Expression levels can be quite high with reports of proteinsexpressed at >20% of the total cell protein, usually as a solubleprotein.

(2) Some proteins cannot be efficiently secreted.

(3) Proteins that contain glycosylation sites, if secreted from yeast,will be glycosylated with the pattern of sugars unique to yeast. Becausethese highly hydrophilic, post-translational modifications lie on thesurface of proteins they are likely to confer antigenicity on theprotein. If the protein is functional without the carbohydrate sidechains it may be advantageous to produce the protein without them ratherthan with the yeast-specific modification (see for ex. Travis et al.1985. J Biol Chem 260:4384-4389).

(4) Some proteins have other specific modifications that may occur onlyin the cytoplasm, for example aminoterminal acetylation, modificationwith lipids, perhaps heme incorporation and so forth.

At present, cytoplasmic expression is preferred since the yeast cellsfold together the globin chains and incorporate heme to producehemoglobin in vivo. However, it is possible to separately express andsecrete the alpha and beta globin chains and assemble hemoglobin invitro.

The globin genes must be placed under the control of a suitablepromoter. The commonly used yeast promoters generally fall into twobroad categories: regulated and constitutive. Constitutive promotersthat are in wide use include GAP, PGK (phosphoglycerate kinase) and theα-factor promoter. Regulated promoters have also been used and theseinclude the yeast metallothionein promoter (regulated by copper), theGal1-10 promoter, GAL7 promoter (regulated by galactose and glucose) theADHII promoter (regulated by ethanol and glucose) the PH05 promoter(phosphate regulation) and several hybrid promoters such as PH05-GAP,GAL-PGK, ADHII-GAP, and GAL-CYC1.

The use of regulated promoters may be important for plasmid stability.Often expression of recombinant proteins, at high levels, inhibits thegrowth of the host organism. This disadvantage can result in theaccumulation of cells in the population with few copies of the plasmidpresent, a decrease in growth rate and a drop in overall expressionlevels. By maintaining the gene in a repressed state and inducing latein the fermentation, high growth rates can be maintained and selectionagainst high plasmid copy numbers can be avoided.

It is somewhat difficult to obtain accurate data on the relativestrength of yeast promoters because the only true measure of this wouldbe based on kinetics of mRNA synthesis. Most of the data that existsmeasures only the final protein concentration that has been obtained.Because there can be great differences in protein stability, it isnecessary to compare the same protein and mRNA with multiple promoterson identical vectors. A study that approaches this was done by Verbakelet al. (Gene, 61:207-215 (1987)). They compared the GAPDH, CYC1 GAL7,PHO5, and PGK promoters and measured the expression of aβ-galactosidase/poliovirus VP2 protein fusion. CYC1 resulted in anexpression level of 0.14% of the total cell protein (TCP); GAPDH, 0.22%TCP; PHO5, 0.26% TCP; PGK, 0.9% TCP; and GAL7, 0.96% TCP.

Using our GALGAP hybrid promoter, we have obtained expression levels ofhemoglobin that represents ≧15% of the total cell protein. This mostprobably represents an increased promoter strength along with a highlystable protein product and perhaps mRNA with an extended half life.

The use of a GAL-GAP hybrid promoter is preferred. Both elements (theGAL_(UAS) and the GAP transcriptional initiation site) are wellunderstood. Studies on the mechanisms of transcriptional regulation ofthe GAL regulon have been fairly extensive. The galactose regulonincludes five genes that encode enzymes required for the utilization ofgalactose. Four of these genes (GAL1, GAL7, GAL10, and GAL2) areexpressed only in the presence of galactose. Galactose induction doesnot occur in the presence of glucose unless the yeast strain bears amutation in the REG1 gene. The GAL1, 7, 10 and 2 genes are regulated byat least two other genes, GAL80 and GAL4. The GAL4 gene is atranscriptional activator protein that activates mRNA synthesis from theGAL1, 7, 10 and 2 upstream activator sequences (UAS_(GAL)). AlthoughGAL4 is constitutively expressed, it is functionally silent in theabsence of galactose. Repression of GAL4 activity, in the absence ofgalactose is maintained by the product of the GAL80 gene. The GAL80protein apparently interacts physically with GAL4 to preventtranscriptional activation. Presumably galactose or a galactosederivative prevents this interaction to allow GAL4 mediated induction.

Haploid strains of S. cerevisiae have three different genes encoding theenzyme glyceraldehyde-3-phosphate dehydrogenase (GAP). These genes havebeen designated TDH1, TDH2 and TDH3 and each is present as a single copyper haploid genome. The TDH3 gene produces approximately 60% of thecell's GAP enzyme and TDH1 and 2 produce about 12% and 28%, respectively(McAllister, L and M. J. Holland, 1985. J. Biol Chem, 260: 15019-15027).Holland's group (Holland et al. 1981. J. Biol Chem, 256:1385-1395; andHolland et al. 1983. J Biol Chem 258:5291-5299) has cloned andcharacterized the three GAP genes of S. cerevisiae. The clones have beendesignated pGAP11, pGAP63, and pGAP491. pGAP491 corresponds to the TDH3gene and is therefore, the most highly expressed. This promoter has beenused to express a wide variety of proteins in yeast including:

    ______________________________________                                        Protein          REF                                                          ______________________________________                                        α Interferon                                                                             (1)                                                          Hepatitis B antigen                                                                            (1)                                                          Thaumatin        (2)                                                          Hepatitis B antigen                                                                            (3)                                                          HIV III reverse  (4)                                                          transcriptase                                                                 human SOC        (5)                                                          α1 antiprotease                                                                          (6)                                                          ______________________________________                                         (1) Bitter, G. and K. M. Eagan. 1984. Gene, 32.263-274.                       (2) Edens, L. et al. 1984. Cell, 37:629-633.                                  (3) Kitano, K. et al. 1987. Biotechnology 5:281-283.                          (4) Hallewell, R.A. et al. 1987. Biotechnology 5:363-366.                     (5) Barr, P.J. et al. 1987. Biotechnology 5.486-489.                          (6) Travis, J. et al. 1985. J Biol Chem 260:4384-4389.                   

This promoter is commonly used as a 600-850 bp fragment and isessentially un-regulated. In its long form this is a very powerfulpromoter. The form we are using consists of only .sup.˜ 200 bp 5' of thetranslational initiation site. This form, with no added enhancersequences is substantially less active than the longer form of thepromoter (Edens, L. et al. Cell, 37:629 (1984)). Our addition of the GALenhancer region confers both regulation and high levels of expression.With only the GAP491 promoter, alpha and beta globin were produced at alevel of less than 0.2% total cell protein; with the GAL-GAP491 hybridpromoter, expression jumped to 7-10% total cell protein.

Several other hybrid promoters are of particular interest:

GAL-SIGMA A strong galactose-regulated promoter with the sigmatranscriptional start site.

SIGMA-GAP A strong peptide hormone-regulated promoter with the GAP491transcriptional start site.

GAL-EF III A strong galactose-regulated promoter with the elongationfactor III transcriptional start site.

SIGMA-EF III A strong peptide hormone-regulated promoter with theelongation factor III transcriptional start site.

One could easily conceive of other promoter systems that would alsowork. This would include, but not be limited to, a variety ofconstitutive promoters. For example, the yeast mating factors (MFα)promoter or the mating factor a promoter MF(a), the phosphoglyceratekinase promoter (PGK), hexokinase1, hexokinase2, glucokinase, pyruvatekinase, triose phosphate isomerase, phosphoglycerate isomerase,phosphoglycerate mutase, phosphofructose kinase or aldolase promotersmay all be used. In short, any well expressed yeast promoter may workfor expression of hemoglobin in yeast. A wide variety of naturallyoccurring, regulated promoters could also be used, for example: GAL1-10,GAL7, PHO5, ADHII have all been used to produce heterologous proteins inyeast. A variety of synthetic or semi-synthetic yeast promoters couldalso be employed such as GAL-PGK, GAL-MFα-1, GAL-MFa1, GAL-SIGMA. ADHIIregulatory sequences could also be coupled to strong transcriptionalinitiation sites derived from a variety of promoters. The PHO5regulatory sequence or the sigma element regulatory sequences could alsobe used to construct powerful hybrid promoters. In addition to yeastpromoters, it is conceivable that one could use a powerful prokaryoticpromoter like the T7 promoter. In this case, one could place the T7polymerase under the control of a tightly regulated yeast promoter.Induction of the phage polymerase in yeast cells bearing hemoglobingenes under T7 promoter regulation would allow transcription of thegenes by this very efficient phage polymerase.

Because most of the yeast regulatory sequences described above serve astargets for proteins that are positive regulators of transcription, itis conceivable that these proteins may limit transcription in situationswhere the target sequence is present in many copies. Such a situationmay obtain with vectors such as pC1B, pCIT, pC1U or pC1N which may bepresent in excess of 200 copies per cell. Over-expression of thepositive regulator (for example GAL4) may result in enhanced expression.It is possible to construct a strain in which the GAL4 gene is alteredto remove its promoter and the promoter replaced with the GAL7 orGAL1-10 promoters, both of which are transcribed more efficiently thanthe GAL4 promoter. In this situation, the positive transcriptionalactivator protein GAL4 would be expressed at elevated level at the timehemoglobin expression was induced.

The consensus sequence for higher eukaryotic ribosome binding sites hasbeen defined by Kozack (Cell, 44:283-292 (1986)) to be: G^(AA) _(G)CCAUGG. Deviations from this sequences, particularly at the -3 position(A or G), have a large effect on translation of a particular mRNA.Virtually all highly expressed mammalian genes use this sequence. Highlyexpressed yeast mRNAs, on the other hand, differ from this sequence andinstead use the sequence AAAAAUGU (Cigan and Donahue, Gene, 59:1-18(1987)). The ribosome binding site that we use for expression of the αand β-globins corresponds to the higher eukaryotic ribosome bindingsite. It is within the contemplation of this invention to systematicallyalter this RBS to test the effects of changes that make it more closelyresemble the RBS of yeast. It should be pointed out, however, thatalterations at the -2, -1 and +3 positions, in general, have been foundto only slightly affect translational efficiency in yeast and inmammals.

Intracellular expression of genes in S. cerevisiae is primarily affectedby the strength of the promoter associated with the gene, the plasmidcopy number (for plasmid-borne genes), the transcription terminator, thehost strain, and the codon preference pattern of the gene. Whensecretion of the gene product is desired, the secretion leader sequencebecomes significant. It should be noted that with multicopy plasmids,secretion efficiency may be reduced by strong promoter constructions.Ernst, DNA 5:483-491 (1986).

A variety of extrachromosomally replicating vectors (plasmids) areavailable for transforming yeast cells. The most useful multicopyextrachromosomal yeast vectors are shuttle vectors that use a fulllength 2μ-circle combined with an E. coli plasmid. These vectors carrygenes that allows one to maintain the plasmid in appropriate yeastmutants and antibiotic resistance markers that allow selection in E.coli. Use of the full-length 2μ-circle, in contrast to vectorscontaining only a partial 2μ sequence, generally results in much higherplasmid stability, particularly in yeast strains that have been cured ofendogenous 2μ plasmid. The pC series of vectors described herein arevectors of this type.

Strains could also be constructed in such a way that the GALGAPhemoglobin expression cassettes were integrated into chromosomes byusing yeast integrating vectors. Although the copy number of thehemoglobin genes would be lower than for plasmid vectors, they would bequite stable and perhaps not require selection to be maintained in thehost cell. Yeast integrating vectors include Yip5 (Struhl, et al, PNAS,76:1035-39, 1989), Yip1 (Id.), and pGT6 (Tchumper and Carbon, Gene,10:157-166, 1980). For information on these and other yeast vectors, seePouwels, et al., Cloning Vector, VI-I, et seq. (Elsevier, 1985).

The alpha and beta globin genes may be introduced by separate plasmids,or both upon the same plasmid. The advantage of a single plasmid systemover a double plasmid system is theoretical. It is generally thoughtthat there is an upper limit to the total number of plasmid copies percell. If it is 1000, for example, the two plasmid system could have only500 copies of α-chain plasmid and 500 of the β-chain plasmid. A singleplasmid of 1000 copies per cell would bear 1000 copies of each α- andβ-chain gene. The number of copies may be irrelevant, however, if otherfactors are limiting. In fact, several groups favor using strains thatcontain genes integrated into various chromosomal loci. Such strainsvery stably maintain the foreign gene and do not require special mediato maintain selection for the plasmid.

Highly expressed yeast genes show a very high codon bias. The genesencoding glyceraldehyde-3-phosphate dehydrogenase and ADH-I, forexample, show a 90% bias for a set of 25 codons. Highly expressed yeastgenes (>1% of the total mRNA) have yeast codon bias indices of >0.90.Moderately expressed genes (0.1-0.05% of the total mRNA) have biasindices of 0.6-0.8, and genes expressed at low levels (>0.05% of thetotal cell protein) have a codon bias of 0.10-0.50 (Bennetzen and Hall,J. Biol. Chem., 257:3026-3031 (1982)). The calculated value for thecodons of the human α-globin cDNA is 0.23. A similar value can becalculated for the β-globin cDNA. Because there is a very highcorrelation between the most commonly used codons, it is possible thathemoglobin expression from the human cDNA in yeast may be limited by theavailability of the appropriate tRNA molecules. If this is so, acomplete synthesis of the gene using the most highly favored yeastcodons could improve the expression levels. It is quite possible thatthe greatest negative effect of adverse codon use would be if there wasan abundance of codons used in the cDNA that are represented by lowabundance tRNAs. In such a case, high level expression of hemoglobincould completely drain that pool of tRNA molecules, reducing translationnot only of hemoglobin but of yeast proteins that happen to use thatcodon as well. In the case of the α-globin human cDNA, the most commonlyused leucine codon is CTG (14 of 21), this codon is never used in highlyexpressed yeast genes (Guthrie and Abelson, The Molecular Biology of theYeast Saccharomyces, Eds. Stratern, Jones and Broach, 1982. Cold SpringHarbor, N.Y.). The low codon bias index and the presence of rare yeastcodons in the globin cDNAs have been sufficient incentive for us tosynthesize a modified form of the alpha- and beta-globin genes using thepreferred yeast codons.

Pseudodimer-Containing Hemoglobins

Although the assembly of secreted F_(v) antibody fragments in which theV_(H) and V_(L) domains are fused by a 16 amino acid peptide has beendemonstrated in E. coli, Better, J., Chang, P. Robinson, R., Horwitz,R., Science 240, 1041-43 (1988), Skerra, A. and Pluckthorn, A., Science240, 1038-41 (1988), there are no previous examples of the cytoplasmicfolding and assembly of a mammalian heterotetramer in which two of thesubunits are fused to one another.

The present invention further contemplates in some embodiments thecombination of (a) one molecule of a di-alpha globin-like polypeptidewith two molecules of a beta globin-like polypeptide to form a"di-alpha" hemoglobin-like protein; (b) two molecules of analpha-globin-like polypeptide with one molecule of a di-beta globin-likepolypeptide to form a "di-beta" hemoglobin-like protein; or (c) onemolecule of a di-alpha globin-like polypeptide with one molecule of adi-beta globin-like polypeptide to form a "di-alpha/di-beta"hemoglobin-like protein.

It should further be noted that the delta, gamma and epsilon chains haveconsiderable homology with the beta chain and that the zeta chain hasconsiderable homology with the alpha chain. Di-delta, di-gamma,di-epsilon and di-zeta polypeptides are therefore within the compass ofthe invention and may be used in the preparation of novel hemoglobins oftypes other than Hgb A1.

In the liganded form, hemoglobin readily dissociates into αβ dimerswhich are small enough to pass through the renal glomeruli, andhemoglobin is thereby rapidly removed from the circulatory system.Intravenous administration of hemoglobin in amounts far less than thatneeded to support oxygen transport can result in long term kidney damageor failure. Ackers, G. K. and Halvorson, H. R., Proc. Nat. Acad. Sci.(U.S.A.) 71, 4312-16 (1974); Bunn, H. F., Jandl, J., J. Exp. Med. 129,925-34 (1969). If dissociation into dimers is prevented, there is anincrease in intravascular half life and a substantial reduction of renaltoxicity. Lee, R., Atsumi, N., Jackbs, E., Austen, W., Vlahakes, G., J.Surg. Res. 47, 407-11 (1989). The pseudotetrameric hemoglobin of thisinvention cannot dissociate into αβ-dimers without the breakage of apeptide bond and should have the advantages of a longer intravascularhalf life and reduce renal toxicity.

In the crystal structures of both deoxyhemoglobin and oxyhemoglobin theN-terminal Val residue for one α subunit and the C-terminal Arg residueof the other α subunit are only between 2 and 6 Å apart, and are boundto one another through a salt bridge in deoxyhemoglobin. Fermi, G.,Perutz, M., Shaanan, B., Fourme, R., J. Mol. Biol., 175, 159-74 (1984);Shaanan, B., J. Mol. Biol. 171, 31-59 (1983). This distance could bespanned by one or two amino acids. One extra amino acid can be added tothe C-terminal Arg residue of the α subunits by trypsin catalyzedreverse hydrolysis without significantly altering the oxygen bindingproperties. Nagai, K., Enoki, Y., Tomita, S. and Teshima, T., J. Biol.Chem., 257, 1622-25 (1982) Preferably the di-alpha linker (if one isused) consists of 1 to 5 amino acids which may be the same or different.A Mono-Gly linker is especially preferred. In designing such a linker,it is important to recognize that it is desirable to use one or moreamino acids that will flexibly connect the two subunits, transformingthem into domains of a single di-alpha globin polypeptide.

The preparation of "di-beta" mutants is also contemplated. The distancebetween the N-terminus of one beta subunit and the C-terminus of theother is 18.4 Å in the deoxy configuration and 5.2 Å in the oxy form.Preferably, the di-beta linker consists of 2 to 9, amino acids which maybe the same or different. Glycine amino acids are particularlypreferred.

The length of the (-gly-)_(n) genetically fused link between theN-terminus of one beta chain (at beta₁, 1 Val) and the C terminus of thesecond beta chain (beta₂, 146 His) in di-beta hemoglobin may rangebetween 1 and approximately 9 glycines. In the oxy and deoxy crystalstructures of human hemoglobin A₀, the distance between these termini is5.22 Å and 17.93 Å respectively (from the N-terminal nitrogen to the Cterminal carbon of the carboxylate). A single glycine linker, which is alittle less than 4 Å in length, may come close to linking the twotermini in the oxy structure, however, it is expected that this linkerwill fall .sup.˜ 14 Å short in the deoxy structure. Significantly moreperturbation of the deoxy structure vs the oxy structure might beanticipated with this linker. Some alterations in the oxygen bindingproperties may be caused by deletion of the positive and negativecharges at the two termini and their inclusion in the amide bond. Inaddition, the linker molecule itself may destabilize the oxy structureless than the deoxy structure, and thus lead to a relative increase inoxygen affinity. Likewise, two glycines inserted as linkers may alsodifferentially stabilize the oxy structure and hence relatively increasethe oxygen affinity by the same mechanism described above.

When the number of linking glycines is increased to 5, the linker shouldjust span the cleft between the beta chain termini in the deoxystructure, and, moreover, insert added steric bulk between the terminiin the oxy structure, thus leading to a relative stabilization of deoxy(or destabilization of oxy) and perhaps resulting in a concomitantdecrease in oxygen affinity. Due to the large space between the betatermini in the deoxy (but not the oxy structure), addition of glycinelinkers in the range of 6-9 may further destabilize the oxy structureand, in the same manner, further decrease oxygen affinity.

A third form of globin pseudodimer is one comprising both alpha and betaglobin domains. A possible route to fusing alpha1 to beta2 and sostabilizing hemoglobin against α₁ β₁ /α₂ β₂ dimer formation, is to fusethe alpha1 C-terminal residue to the N-terminal residue of beta2 Chelix, creating a new C-terminus at the end of the beta2 B helix. Theoriginal beta N terminus, Val1, would be fused to the original betasubunit C-terminal residue, His146, by means of an intervening newsection of protein, thus creating a continuous polypeptide chaincomprising the alpha and. beta subunits of different dimers. This chainmay be described as follows: α(1-14)-Gly₃ -β(35-146)-Gly₁₋₃ -Ala₁₃-Gly₁₋₃ -β(1-34); See FIG. 36.

Inspection of the structure of human deoxyhemoglobin using. a moleculargraphics computer indicates the following relevant distances. Thedistance between the Alpha1 Arg141 carboxyl carbon and Beta2 Tyr35 Natoms is approximately 8.5 Angstroms. A fully extended linear triglycinepeptide measured approximately 10.1 Angstroms from the N to C terminalresidues. This suggests that three glycine residues could be employed tospan the distance between the Arg141 and Tyr35 residues with a minimumof unfavorable steric interactions and maximum conformational freedom.The distance requirements could be different in oxyhemoglobin, and ifso, the sequence of the fusion peptide could be altered to bestaccommodate the requirements of both structures.

In human deoxyhemoglobin, the distance between the Beta2 His146 carboxylcarbon and the Beta2 Val1 nitrogen atoms is approximately 25 Angstroms.A right handed 3.6 Alpha helix constructed from a linear sequence of 13Alanine residues was found to measure 22 Angstroms from N to C terminus.With the addition of one to three glycine residues at each end of thishelix (to give Gly_(n) (Ala)₁₃ Gly_(n) where n=1 to 3), it could spanthe required distance and have sufficient conformational flexibility toavoid serious tertiary packing conflicts. Additionally, the amino acidsequence of the helix could be altered to introduce favorable hydrogenbonds and salt bridges between the new helix and the Beta2 helix againstwhich it would pack in the folded protein. Such interactions could aidstabilization of the engineered protein.

Glycine is the preferred amino acid in the linkers, since it is known tobe quite flexible, Cantor and Schimmel, Biophysical Chemistry, part 1,pp. 266-9 (1980), and also allows chains into which it is incorporatedto assume a more compact structure. However, the residues comprising thelinker are not limited to glycines; other residues may be includedinstead of or in addition to glycine, such as alanine, serine, orthreonine. Since these amino acids have a more restricted conformationalspace in a protein, they will likely result in more rigid linkingchains, and hence have a more pronounced effect on the relativestabilization/destabilization of the oxy/deoxy structures.

It should be understood that the minimum and maximum number of aminoacids in the linker is a function of the distance to be spanned in boththe oxy or deoxy forms, the amino acids chosen, and the propensity ofthe particular amino acid sequence to form a secondary structure. Whilea random coil is usually preferred, it is not required, and a linkerwith a larger number of amino acids in a secondary structure may havethe same span as a random coil linker with fewer amino acids. A linkermay comprise, e.g., 1-3 glycines, followed by a sequence having asecondary structure, followed by 1-3 more glycines. The translation perresidue, in angstroms is 1.9 for polyproline I, 3.12 for polyproline II,3.1 for polyglycine II, 3.4 for an antiparallel β sheet, 3.2 for aparallel β-sheet, 1.5 for a right handed a-helix, 2.0 for a 310 helix,and 1.15 for a π helix. In a fully extended chain, the maximumtranslation per residue is 3.63 Å if the repeating units are staggeredand 3.8 Å if the peptide bond is trans.

The number of amino acids in the linker may be such that a formation ofa secondary structure, such as an alpha helix or a beta-sheet, isundesirable, as the span is reduced. Certain amino acids have a greatertendency to participate in such structures. See Chou and Fasman,Biochemistry, 13:222-245 (1974), incorporated by reference. The aminoacids are ranked in order of decreasing participation below. Thepreferred linker amino acids are boldfaced. Glycine is the most suitableamino acid for this purpose. The most preferred di-alpha linkers are Glyor Gly-Gly.

    ______________________________________                                        Alpha Helix         Beta Sheet                                                Formers             Formers                                                   ______________________________________                                        Glu (1.53)          Met (1.67)                                                Ala (1.45)          Val (1.65)                                                Leu (1.34) Hα Ile (1.60) Hβ                                        His (1.24)          Cys (1.30)                                                Met (1.20)          Tyr (1.29)                                                Gln (1.17)          Phe (1.28)                                                Val (1.14)          Gln (1.23)                                                Trp (1.14)          Leu (1.22)                                                Phe (1.12) hα Thr (1.20)                                                Lys (1.07)          Trp (1.19) hβ                                        Ile (1.00)          Ala (0.97) Iβ                                        Asp (0.98)          Arg (0.90)                                                Thr (0.82)          Gly (0.81)                                                Arg (0.79)          Asp (0.80) iβ                                        Ser (0.79)          Lys (0.74)                                                Cys (0.77) iα Ser (0.72)                                                Asn (0.73)          His (0.71)                                                Tyr (0.61) bα Asn (0.65)                                                Pro (0.59)          Pro (0.62) bβ                                        Gly (0.53) Bα Glu (0.26) Bβ                                        ______________________________________                                    

(The letter symbols are Hα, strong α former; hα, α former; Iα; weak αformer; iα, α indifferent; bα, α breaker; and Bα strong α breaker. The βsymbols are analogous. Trp is bβ if near the C-terminal of a β-sheetregion.)

The alpha helix of a polypeptide chain comprises an average of 3.6residues per turn. In globular proteins, the average length is about 17Å, corresponding to 11 residues or 3 helix turns. In alpha and betaglobin, the helices range in length from 7 to 21 amino acids (A.A.). Thebeta pleated sheet comprises 2.3 residues per turn; the average lengthis about 20 Å or 6 residues.

Chou and Fasman define an alpha helix nucleus as a hexapeptidecontaining four helix forming residues and not more than one helixbreaker, and a beta sheet nucleus as a pentapeptide containing threebeta sheet forming residues and not more than one sheet breaker.

The amino acid sequence in the vicinity of the di-alpha linker is asfollows:

    __________________________________________________________________________    residue #                                                                          138 139                                                                              140                                                                              141      1  2   3  4                                           AA   Ser Lys                                                                              Tyr                                                                              Arg                                                            (XXX).sub.n -                                                                      Val Leu                                                                              Ser                                                                              Pro                                                            Helix Not                                                                          H21 HC1                                                                              HC2                                                                              HC3      NA1                                                                              NA2 A1 A2                                          Helix Pot                                                                          079 107                                                                              061                                                                              079      114                                                                              134 079                                                                              059                                         Sheet Pot                                                                          072 074                                                                              129                                                                              090      165                                                                              122 072                                                                              062                                         __________________________________________________________________________

(Note: Helix- and sheet forming potentials have been multiplied by 100for typographical reasons.)

The di-alpha linker is preferably only 1-3 amino acids. Thus, it canform an alpha helix only in conjunction with the linker "termini". A oneor two residue linker, even if composed of amino acids with strongsecondary structure propensities, would be unlikely to assume an alphahelix or beta sheet configuration in view of the disruptive effect of,e.g., Arg 141 or Ser 3. If the linker is 3 residues long, it would bepreferable that no more than one residue be a strong alpha helix former,unless the linker also included a strong alpha helix breaker.

The amino acid sequence in the vicinity of the di-beta linker may imposemore stringent constraints.

    ______________________________________                                        143  144     145    146         1    2     3    4                             His  Lys     Tyr    His                                                       (XXX).sub.n -                                                                      Val     His    Leu  Thr                                                  H21  HC1     HC2    HC3         NA1  NA2   NA3  A1                            124  107     061    124         114  124   134  082                           071  074     129    07:L        165  071   122  120                           ______________________________________                                    

The di-beta linker is likely to be longer (preferably 1-9 A.A.) andtherefore more susceptible to secondary structure formation. Ifsecondary structure formation is not desired, it is desirable that theamino acid adjacent to Val-1 be an alpha helix breaker (e.g., Glycine)in view of alpha-helix propensities of Val-His-Leu. More generally, itis desirable that the linker not contain (or cooperate with theproximately linked amino acids to form) an alpha helix nucleus or betasheet nucleus.

When secondary structure is not desired, amino acids with a highpropensity toward alpha helix formation may be used in the linker ifaccompanied by "helix breaking" amino acids. Similarly, Beta sheetformation may be prevented by "sheet disrupting" amino acids.

Of course, prediction of secondary structure using Chou and Fasman'sapproach has its limitations and the ultimate test of the acceptabilityof a linker is whether or not the di-alpha or di-beta hemoglobin has thedesired affinity for oxygen. In particular, a poly-alanine linker,despite its supposed propensity to alpha-helix formation, may well be ofvalue since the alanine group is compact and therefore the linker shouldbe quite flexible if secondary structure does not form.

In an especially preferred embodiment, di-alpha and beta globin genesare combined into a single polycistronic operon. The use of apolycistronic operon is not, however, necessary to practice the presentinvention, and the alpha (or di-alpha) and beta (or di-beta) globingenes may be expressed from separate promoters which may be the same ordifferent.

While the preferred "genetically fused hemoglobin" of the presentinvention is one comprising a di-alpha and/or di-beta globin, otherglobin chains may be genetically fused and used in the production ofhemoglobins of species other than Hgb A1 (α₂ β₂).

Pseudo-Octameric (Ditetrameric) Hemoglobin-like Proteins With DisulfideBridges

The ability to produce pseudotetrameric recombinant hemoglobinsconsisting of a single dialpha polypeptide and two beta chains (or adibeta polypeptide and two alpha chains) provides a unique opportunityto create an asymmetric pseudotetramer from the normally symmetricpseudotetramer. Because the two alpha globin domains are expressed as asingle polypeptide, it is possible to alter one of the alpha globindomains without altering the other. The result is a protein that, in itsfinal folded state, contains two different alpha globin domains in astrict 1:1 ratio. This type of asymmetric hemoglobin molecule, with itsunique chemical properties, cannot be easily constructed by any othermethod. A preferred embodiment of this invention would involve use ofsite-directed mutagenesis to substitute a cysteine residue in one of thetwo alpha globin domains of a di-alpha hemoglobin such as SGE1.1 (adi-alpha hemoglobin with a beta chain Presbyterian mutation) such thatthe cysteine would be on the surface of the folded recombinanthemoglobin molecule. A homogeneous preparation of pseudo-octamerichemoglobin could then be formed through interhemoglobin linkage of twopseudotetramers either directly by simple oxidation of purifiedpseudotetramers or by reaction with a bridging molecule (FIG. 27).

Advantages of a pseudo-octamer stem from several possible therapeuticfeatures of this construct: (I) half-life in the bloodstream of thepseudo-octamer (MW.sup.˜ 128,000) is expected to be longer than thehalflife of pseudotetrameric Hb (MW.sup.˜ 64,000). Studies of halflifeas a function of macromolecular size indicate a correlation betweenincreased size and increased circulatory halflife for chemicallycrosslinked Hb as well as other macromolecules. (II) because the numberof oxygen binding heme groups per octamer is twice the number pertetramers, independent of size, the oncotic pressure for a givenconcentration of heme groups in a solution of octameric Hb is expectedto be half that of an equimolar solution of heme contained in tetramericHb. Because of oncotic pressure effects, the maximum concentration offree tetrameric Hb that may be introduced into the blood stream is lesson a per volume basis than the concentration of Hb normally carried inintact red blood cells. Reduction of oncotic pressure is thereforeuseful in increasing the per volume oxygen carrying capacity of a bloodsubstitute. (III) although free hemoglobin purified from natural sourcesmay be polymerized by chemical crosslinking to increase halflife viaincreased molecular weight, and to reduce oncotic pressure, all suchpreparations are heterogeneous. Not only does the genetic engineeringapproach provide a means of synthesizing a higher molecular weightcomplex without the need to involve chemical crosslinking and consequentadditional purification steps, this approach also provides the firstmeans of exerting strict control over the degree of polymerization. Theability to strictly control formation of pseudo-octamers will greatlyfacilitate characterization of the final product and will reduce thechance of adverse reaction to minor components.

Although direct formation of a disulfide bond between two SGE1.1 mon cyshemoglobins is desirable in order to avoid the need for chemicalcrosslinking, the halflife of such a bond in the bloodstream is unknown.Naturally occurring reducing agents may reduce the disulfide bond invivo at a rate comparable to or greater than the halflife of thepseudo-octamer. The rate of reduction of the bond may be influenced bythe location of the cysteine mutation on the surface of the hemoglobin.It may also be possible to insert two cysteine mutations in eachhemoglobin in close enough proximity that steric hindrance would allowformation of a two-disulfide pseudo-octamer but prevent formation ofhigher molecular weight complexes.

Poly(tetrameric) Hemoglobin With Other Intercysteine Linkages

It is also possible, of course, to couple two SGE1.1 mono cys moleculeswith a homobifunctional crosslinking reagent resulting in linkage vianonreduceable bonds. The degree of polymerization is still controlled bythe use of the mono cys di-alpha or di-beta Hgb starting material. (Notethat reference to SGE1.1 mono-cys in this section is for illustrationonly; other mono-cys di-alpha or di-beta Hbgs could be used in itsplace.)

By using bi-, tri-, tetra-, hexa-, or octa-functional crosslinkersseveral properties of multimeric hemoglobin which may contribute tolonger serum half life can be controlled. The crosslinkers can bedesigned to give a nonreducible disulfide bond between two tetramers, toyield high molecular weight multimers of n>2 psuedo-tetramers (e.g.dodecamers, etc.) and/or to drop the overall isoelectric point of ahemoglobin octamer to further increase its half life.

Correlations of molecular weight with serum half life for proteins suchas IL-2, demonstrate that a significantly longer half life may beexpected as the molecular weight of a protein increases, particularlyabove the renal filtration limit of 50-70 kDa. SGE 1.1 mono-cysoctameric hemoglobin will have a molecular weight of ca. 130 kDa, andthis may double or triple its half life. However, a factor potentiallylimiting the half life of octameric hemoglobin formed by a disulfidelink between two mono-cys SGE1.1's is reduction of the cys-cys disulfidebond by endogenous thiol-reducing agents found in the serum. Estimatesof small molecule thiol levels in plasma vary from 17 μM to 5 μM. Themajor species is reduced glutathione. Other thiol compounds in plasmainclude cysteine, homocysteine, and gamma-glutamyl cysteine. Thus, smallmolecule plasma thiols are available for reduction of disulfide bonds.This may be reflected in the diminished half life seen withantibody-ricin A chains conjugates linked by regular disulfides (6.7hrs) relative to conjugates linked with sterically hindered, and thusless reducible, alpha-methyl disulfides (42.5 hours).

Thus, in one embodiment, the octameric hemoglobin features anonreducible disulfide crosslink such as a thioether bond orthiol-maleiimide adduct which may substantially extend the octamer halflife. Simple homobifunctional crosslinkers or polyethylene glycol (peg)derivatives would likely be useful for this purpose (see below). Thereaction of a bifunctional cysteine-specific crosslinker with a mono-cysdi-alpha or di-beta Hgb should limit the products of the reaction tosubstantially a dumbbell-like octameric hemoglobin (FIG. 22) andunreacted hemoglobin. The reaction should be stoichiometric when the Hgband crosslinker are present at high concentrations and the Hgb ispresent in a slight excess over the crosslinker maleiimides at pH6.5-7.0. Further, there should not be substantial interference byreaction with SGE1.1 lysines. The preferential reactivity of the thiolsto lysines can be roughly calculated as the product of their molarratios and the ratio of the intrinsic reactivity of a maleiimide tothiols versus amines. This product is ca. 1 cys/40 lys!× 1000!=25 at pH7. The side products would still be octamers, with one attachment sitebeing a secondary amine and thus might well be functionally equivalentto the S-crosslinked octamers. Hydrolysis of the maleiimide adduct at pH7 would be slow, and the ring opening would leave the crosslink intact.

The following are examples of homobifunctional crosslinkers that mayform metabolically stable crosslinks between monocysteine pseudotetramers:

1) 1,2-bis-(2-iodoethoxy)ethane

2) 4,4'-dimaleiimidylbenzene or N,N'-p-phenylenedimaleiimide

3) N,N'-bis-(3-maleiimido-propionyl)-2-hydroxy-1,3-propane diamine.

Longer half lives may also be obtained by increasing the apparentsolution molecular weight by simply lengthening the distance between thetwo linked tetramers using a long crosslinking agent. The use of somepotentially novel polyethylene glycol derivatives as homobifunctionalcrosslinkers, reacting with SGE1.1 mono-cys, may provide one mechanismfor significantly increasing the molecular weight of octamerichemoglobin by virtue of the length of the crosslinker alone.

A suitable crosslinker for this purpose is maleiimido-CH₂ CH₂ C(═O)(OCH₂CH₂)_(n) OC(═O)CH₂ CH₂ -maleiimido.

The length may be adjusted by variation of n. A few examples are givenbelow.

    ______________________________________                                        Structure Max Length       Source                                             ______________________________________                                        n = 22     .sup.˜ 49 Å                                                                         peg -1000                                          n = 76    .sup.˜ 166 Å                                                                         peg -3350                                          n = 227   .sup.˜ 499 Å                                                                         peg -10000                                         ______________________________________                                    

Homobifunctional N-hydroxysuccinimide-activated peg has been usedpreviously to derivatize hemoglobin. Yabuki, et al., Transfusion, 30:516(1990). This reaction resulted in a mixture of monomeric, dimeric, andtrimeric species with an average stiochiometry of peg/hemoglobin of 6.2.However, 83% of the hemoglobin derivatized by peg was not crosslinked toanother hemoglobin molecule. Control of the peg-derivitization ofwild-type hemoglobin is not possible because there is no site-directedlabeling of the hemoglobin starting material.

In contrast, the combination of SGE 1.1 mono-cys starting material and apeg crosslinker should yield a monodisperse dumbbell (pseudo-octameric)product only. The site-direction of the crosslinker attachment siteshould result in precise control of the apparent molecular weight, whichwill depend on the size of the crosslinker. Moreover, careful control ofthe site of the cys mutation on the surface of the recombinanthemoglobin should ensure that the functionality of the derivatizedhemoglobin is maintained.

Higher Poly(tetrameric) Hemoglobins

The above crosslinkers all involve the attachment of two hemoglobins ateach end of a crosslinker. It may be advantageous to attach more thantwo hemoglobins to a single crosslinker to yield more oxygen-carryingcapacity and to further increase the molecular weight. A hypothetical4-hemoglobin coiled-coil linker complex is shown in FIG. 28(a). Designand synthesis of these coiled coil peptides has already been explored(for an example see Cohen and Perry, Proteins, 7:1-15 (1990)). Therationale for a coiled coil is that two intertwined alpha helices willbe less sensitive to proteolytic cleavage than a single naked secondarystructure like an extended peptide (rapidly cleaved by proteases), analpha helix or a beta sheet.

Using molecular modeling, an internal disulfide may be designed in thecenter of a bi-functional coiled coil linker such that the strands arecovalently attached. This should stabilize formation of the correctcoiled coil crosslinker before mono-cys di-alpha or di-beta Hgb (e.g.,sge1.1 cys) is attached. Additionally, a tri-functional crosslinker canbe stabilized by use of a orthogonally-protected lysine (lys-FMOC)rather than a disulfide in the center of a proteolytically inertsecondary structure. A polyproline helix can be used as the linker, andcan be stabilized by branching the synthesis at the lys-FMOC afterremoval of the side chain. The three remaining lysines in the branchedpeptide would then be iodoacetylated to site-specifically attach athiol-reactive group using either iodoacetic anhydride orN-succinimidyliodo-acetate and subsequently reacted with sge1.1-cys. Ananalogous tetra-functional crosslinker could be synthesized by inserting1-2 prolines between two internal branching lysines to rotate them suchthat the two internal branching chains growing off the orthogonallyprotected lysines head in (nearly) opposite directions. Analogousstructures could be made using D-glutamate(E) or D-aspartate(D) toprovide protease resistance, and these would form an extendedpolyanionic chain at pH 7.

The sequence of a hypothetical alpha-helical coiled coil is modifiedfrom that given in Semchuck, et al., in Peptides: Chemistry, Structureand Biology; 566 (Rivier and Marshall, eds:1990), to leave only twolysines (K) at each end:

AcKCAELEGRLEALEGRLEALEGRLEALEGRLEALEGKL-amide (SEQ ID NO:1)

This coiled coil should have about 10 turns of a helix and thus will beca. 54 Å long, allowing two hemoglobins to attach on each side withoutsteric interference. The exact sequence and length to allow appropriateplacement of 4 sge1.1's would depend on the results of molecularmodeling.

Suggested trifunctional and tetrafunctional crosslinkers are diagrammedbelow. ##STR1##

Another possibility is an 8-hemoglobin complex (FIG. 28(b)). Therationale for considering this sort of complex is that it may be the wayto obtain a very long halflife SGE1.1, due to the extreme stability ofthe "crosslinker" and the substantially higher molecular weight of thecomplex. The crosslinker might take the form of a doubly branched coiledcoil, with a Lys (FMOC) replacing an Arg in the middle of the chain toallow the branching, and with a polyproline helix or other proteaseresistant secondary structure comprising the branching moiety. Thisstructure could allow attachment of 6 SGE1.1's per crosslinker.Alternatively, a 4-helical bundle protein (See FIG. 28(c)) or 4-strandedcoiled-coil such as those synthesized by DeGrado, Science, 243:622(1989), with each helix in the 4-helical bundle containing the consensussequence GELEELLKKLKELLKG (SEQ ID NO:2), the helices being linked bythree PRR or RPR loops, could be utilized as a suitable core for thelinker. This is one of the most stable proteins known, with a G=-22kcal/mole separating the folded from the unfolded state. Each helixwould be 4+ turns or ca. 22 Å long. Since this may not be enough room tofit two hemoglobins with one anchored at each end of the helix, theymight have to be attached to different faces of the same helix, tolysines placed at each end of the polar face of each helix. Each helixis amphipathic; this should allow relative freedom to have a total of 8,lysines (and no more) and to change the remaining lysines to arginines.At least two of the i, i+4 salt bridges per helix would be retained forstability of the protein. Attachment of SGE1.1-SH could be viaiodoacetylation of the lysine epsilon amino groups and then reactionwith SGE1.1-SH.

An example of a modification that might allow more room betweenhemoglobins would be addition of one turn of the helix to the N-terminusof the A and C helices and the C-terminus of the B and D helices. Thisand similar modifications would be subject to modeling and experimentalconstraints.

Analogous core proteins could be made as mutants of known 4-helicalbundle proteins such as myohemerythrin or apoferritin, with the surfaceresidues changed so that 8 (or more if topologically possible) lysines(2 per helix) exist on the surface for subsequent modification andattachment of SGE1.1.

Poly(tetrameric) Hemoglobins with Reduced Isoelectric Points

If the isoelectric point of the whole crosslinked conjugate also affectsthe serum half life, via electrostatic exclusion from the renal filter's"pore", additional negative charges could be included in the crosslinkitself (rather than in the hemoglobin, which could change the functionof the molecule) to drop the isoelectric point of the overallcrosslinked particle. An additional benefit of this might be reduceduptake by the reticuloendothelial system, this uptake being a functionof pI for cationized albumin.

We have preliminary evidence from succinylation of SGE1.1 whichcorrelates the number of modified lysines with isoelectric point. Thisgives a rough estimate of the number of lys to glu and/or lys to aspmutations which may be necessary to reach a pI of 5 or less, the pIrange which we expect we need to significantly extend half life. Webelieve that as many as 8 lysines may have to be modified (a total shiftin charge of 16 units) to drop the pI roughly 2 units. It should be lessdisruptive of the functional properties of hemoglobin to do this via apeptide crosslinker rather than by mutation of the alpha and beta globinsubunits proper. However, some mutations could be made in thecrosslinker and the remainder in the subunits. As before, the SGE1.1-cyswould be attached to iodoacetylated lysine epsilon amino groups byreaction at pH 6.5-7.0.

For human serum albumin in the rat, the half life varied roughlylinearly with the pI of the protein, from ca. 4.6 hours for nativealbumin (pI=4) to 0.8 hrs at a pI above 9.5. Clearance was probably bymultiple mechanisms, including potentially increased uptake into thereticuloendothelial system with increased pI. For rat trypsinogens, thedifference in serum half life between versions with a pI of 5.0 (t_(1/2)of 4 min) was even larger. Thus a lower pI clearly appears to be animportant variable in the serum half life of these proteins.

The following table gives examples of crosslinkers between SGE1.1-cystetramers which should diminish the isoelectric point of the overallcomplex.

    ______________________________________                                        Source  Sequence                                                              ______________________________________                                         ##STR2##                                                                              ##STR3##                                                                                        ##STR4##                                            ##STR5##                                                                              ##STR6##                                                                                        ##STR7##                                            ##STR8##                                                                              ##STR9##                                                                                        ##STR10##                                          ______________________________________                                    

A number of the proposed crosslinkers could combine at least two, orpossibly three of these attributes for potential additive effects.

It is possible that the unique amine groups in the peptide crosslinkerscould be directly iodoacetylated during the peptide synthesis bytreating iodoacetic acid as the last amino acid to be added, afterdeprotecting the lysine amine groups on the resin. In this case, thelysines would be orthogonally protected with N-FMOC orN-nitropyridinesulfenyl groups, or with BNPEOC. This could greatlysimplify their synthesis.

Alternate methodologies to iodoacetylation as part of the synthesiscould include the reaction of either sge1.1-SH or the peptidecrosslinker with a heterobifunctional crosslinker specific forsulfhydryls and amines, such as sulfo-SMCC or similar reagents availablefrom Pierce Chemical Co. (Rockford, Ill.).

Genetically Fused Poly(tetrameric) Hemoglobins

Another approach to the preparation of multimeric (e.g., polytetrameric)hemoglobin involves the genetic fusing of individual tetramers utilizingother linkers. Two or more tetramers may be crosslinked, depending onthe desired molecular weight and the efficiency of folding of the finalmolecule. The brine shrimp Artemia produces three polymeric hemoglobinswith nine genetically fused globin subunits. Manning, et al., Nature,348:653 (1990). These are formed by variable association of twodifferent subunit types, a and bβ Of the eight intersubunit linkers sixare 12 residues long, one is 11 residues and one is of 14 residues.Similarly, the dialpha (or dibeta) subunits from different tetramers ofa di-alpha or di-beta Hgb might be genetically fused together into anextended polypeptide which would link the individual pseudotetramericdomains.

Proteolytically stable extended polypeptide linkages can be envisioned.Desirable linker features might include 1) a number of glycines at eachend to allow flexibility in entering the dialpha (or beta) terminaldomains, and to decouple the linker secondary structure from that of thedialpha (or beta) terminal domains; 2) stiffness to separate tetramers,obtainable by an extended structure such as a polyproline helix or bypolyglutamate or polyaspartate; and 3) inertness to proteases (videsupra or as in a collagen sequence). Several examples of such sequencesare listed below. Obviously any other of the peptide linkers mentionedin this specification could be tried after first sterically modeling thefused-dialpha (or dibeta) termini environment. The links would go fromthe C-terminus of one dialpha to the N-terminus of the next and would besynthesized as a single gene. Besides modeling segments ofprotease-resistant or negatively charged secondary structure, one ormore of the Artemia linkers should be modeled between tetramers. Thebeta chains could also be joined in this fashion, although the resultsof this on protein function would be unknown. It might be feasible tomake an intermolecular di-beta (sge1.1) with or without additionalintrachain crosslinkages.

    __________________________________________________________________________    Source   Sequence                                                             __________________________________________________________________________    polyproline helix                                                                      di α or β C term-(G)n-(P)n-(G)n-di α or β               N terminus                                                                    n probably ≧ 3, m probably ≧10-12                      polyaspartate or -                                                            (G)n-(D)n-(G)n- (should drop pI of complex)                                   glutamate                                                                     Artemia linker                                                                (G)n-LRRQIDLEVTGL-(G)n- ; n ≧ 0 (SEQ ID NO:3)                          (example)                                                                     a helical coiled coil                                                         (G)n-KCAELEG(KLEALEG).sub.4 <- not fused to terminus (SEQ ID NO:4)                     (should form octamer with coiled-coil crosslink)                     __________________________________________________________________________

Two structures of human hemoglobin A₀ (either both in the oxy form orboth in the deoxy form) taken or assembled from the Brookhaven ProteinData Bank were docked as close together as possible without van derWaals overlap between any residues, using the program Insight (Biosym.Inc., San Diego, Calif.). The distance from the alpha chain C terminalresidue arg 141 to the amino terminal nitrogen of the alpha chain Nterminal residue val 1 (in one structure) was then measured. Thisdistance was ca. 22 Å when both molecules had the oxy structure and ca.18 Å when both were in the deoxy structure. In the oxy and deoxystructures, the valine at the alpha chain N terminus is exposed at theside of a cleft in the structure, while the arg carboxylate is at thebottom of the cleft. Thus it is possible to genetically fuse these twotermini without suffering a large structural displacement of residues around either terminal amino acid. Linkers which have sufficient lengthto stretch between these termini when the two hemoglobins are dockedtogether (but without any van der Waals overlap) are listed below.

An alternative fusion may be envisioned between a truncated alpha chainin one hemoglobin and the N terminal alpha val 1 in the secondhemoglobin. The first molecule could be truncated at ser 138, whichintermolecular N terminal to C terminal distance is about 17 Å (deoxy)and 22 Å (oxy), and examples of genetically inserted linkers spanningthis distance are listed below.

Thus two hemoglobin molecules could be linked (by fusing twointermolecular alpha domains) to generate a fusion protein approximatelytwice the size of normal human hemoglobin. An additional intramolecularcrosslink, as introduced into rHb1.1 to prevent dissociation ofhemoglobin into dimers, could be included as well, giving a fusion offour alpha domains.

We expect that the genetically inserted links will be stable in thepresence of proteases, due to the steric occlusion by the twohemoglobins surrounding the linkage. This resistance may be furtherenhanced by the use of glycines, bonds between which may be lesssusceptible to proteases, since most proteases have side chainspecificity for residues other than glycine (which has only a hydrogenas a sidechain, and thus may result in a poor Km of this substrate for aprotease). A polyproline helix may also be used as a linker to enhancestability to proteases. Fusion of a polyglutamate or polyaspartate as alinker might allow a much lower isoelectric point for the complex, andthus a longer serum half life.

    __________________________________________________________________________                  end-to-end                                                                          con-                                                      linker        Distance                                                                            formation                                                                            comments                                           __________________________________________________________________________    (gly).sub.7 - 25 Å                                                                            extended                                                                             minimal length for                                                            gly linker to span                                                            termini in both                                                               oxy and deoxy                                                                 structures.                                                                   Longer linkers (up                                                            to 20-50 residues)                                                            may also work                                                                 favorably.                                         (gly).sub.1-3 -(ala).sub.12 -(gly).sub.1-3 -                                                20 Å-40 Å                                                                   Ala in the Gly are added                                                      right  for flexibility                                                        handed and minimal                                                            alpha  disturbance of Hb                                                      helix  structure around                                                              their fusion with                                                             the N and C                                                                   termini. Length                                                               is dependent on                                                               the number of                                                                 glycines and the                                                              degree of                                                                     extension.                                         (gly).sub.1-3 -(pro).sub.12-16 -(gly).sub.1-3 -                                             21-48 Å                                                                         pro in a left                                                                        12, 14, 16                                                             handed poly-                                                                         prolines. Length                                                       proline helix                                                                        dependent on                                                                  number of prolines                                                            and glycines                                       (gly).sub.1-3 -(asp).sub.n -(gly.sub.1-3)-                                                  26-49 Å  Asp residues add                                                              negative charges                                                              in pseudooctamer.                                  __________________________________________________________________________

Other residues could be substituted into these linkers while leavingtheir length essentially the same, including complete linkers taken fromthe sequence of other known human proteins such as hemoglobin, toprevent any recognition of the pseudooctamer as a foreign protein.

Use of linkers with a maximal length more than 18 Å and less than 22 Åmay differentially stabilize the deoxy structure, and may result in alowered oxygen affinity for the pseudooctamer.

Octameric Hemoglobins Formed Without Use of an Pseudooligomeric Globin

It is possible to produce an octameric hemoglobin, without substantialproduction of higher multimers, by suitable cysteine mutation of eitherthe alpha or beta chain (see FIG. 35).

Hemoglobin mutants containing one X to cys mutation in the beta chaingene (giving two per tetramer) or in the alpha chain gene (also givingtwo per tetramer), in which the residues mutated to cysteine are both onor very close to the surface of the subunit and are as close (preferably10-15 Å) to the dyad axis separating the subunits, may form octamers(two hemoglobins) linked by two disulfides. Polymerization of suchmutants should be retarded by the proximity or the two disulfides toeach other, such that after one disulfide is formed, a third incominghemoglobin will be sterically hindered from reacting with either freecysteine on the two original hemoglobins.

Because it is possible that this mutant may form higher order polymers(rather than simply the octamer), a diluted solution may be used invitro for formation of disulfide bonds. The kinetics of polymerizationof hemoglobin should be at least second order (or a higher order) inhemoglobin concentration, while after one disulfide is formed, theformation of the second disulfide between two tetramers should be zeroorder in hemoglobin. Thus the ratio of polymerized product to octamershould diminish as the hemoglobin concentration is decreased. Ifformation of octamers is done under oxygenated conditions, the yield ofoctamers vs. polymers may increase further, since the distance betweenthe two cys mutations is less in every case in the oxy hemoglobinstructure than in the deoxy structure.

A list of preferred mutation sites in both the beta chain and the alphachain is provided below:

Beta and alpha chain mutation sites for x to cys mutations to formdisulfide-bond linked octameric hemoglobin.

    ______________________________________                                        Chain  mutation   distance                                                                              distance,                                                                             Comment                                     ______________________________________                                        beta asn                                                                             80 to cys  22 Å                                                                              18 Å                                                                              no listed                                                                     deleterious                                                                   mutations, asn 80                                                             is on surface.sup.a                         beta asp                                                                             79 to cys  24 Å                                                                              22 Å                                                                              Hb Tampa.sup.a (asp to                                                        tyr) has no major                                                             abnormal property                                                             listed; Hb G-Hsi-                                                             Tsou (asp to gly)                                                             has increased O.sub.2                                                         affinity; is on                                                               surface                                     alpha asn                                                                            78 to cys  24 Å                                                                              20 Å                                                                              on surface; no                                                                major.sup.a abnormal                                                          properties of                                                                 known mutations of                                                            asn 78                                      alpha asp                                                                            75 to cys  22 Å                                                                              18 Å                                                                              on surface; no                                                                major.sup.a abnormal                                                          properties of                                                                 known mutations of                                                            asp 75                                      alpha asp                                                                            74 to cys  26 Å                                                                              20 Å                                                                              on surface; no                                                                major.sup.a abnormal                                                          properties of                                                                 known mutations of                                                            asp 74                                      ______________________________________                                         .sup.a R. N. Wrightstone. Policies of the International Hemoglobin            Information Center (IHIC), Comprehensive Sickle Cell Center, Medical          College of Georgia. 1988.                                                

Miscellaneous

The appended claims are hereby incorporated by reference as a furtherenumeration of the preferred embodiments. All cited references areincorporated by reference to the extent necessary to enable the practiceof the invention as now or hereafter claimed.

By the means suggested in this specification, we have had the followingachievements:

(1) Expression of Met-FX-alpha globin in E. coli from a dicistronicoperon comprising a introductory cistron and a Met-FX-alpha globin gene,both transcribed from a Tac promoter. (Example 2)

(2) Expression of Met-FX beta globin in E. coli by similar means.(Example 2)

(3) Co-expression of Met-FX-alpha globin and Met-FX-beta globin in E.coli from a tetracistronic operon comprising an introductory cistron, anFX-alpha globin gene, an intercistronic sequence, a second introductorycistron, and an FX-beta globin gene, all controlled by a single Tacpromoter. The alpha and beta globins were intracellularly assembled intofunctional FX hemoglobin. The FX Hgb was enzymatically converted to Hgb.(Example 3)

(4) Co-expression as in (3) above of mutants of FX-hemoglobin in whichthe beta globin subunits possessed the Beth Israel, Cheverly,Providence/MSR, Kansas or beta⁶⁷ Val→Ile mutations. (Example 4)

(5) Co-expression as in (3) above of Met-alpha globin and Met-betaglobin and intracellular assembly into Met-Hgb (a.k.a. Des-FX-Hgb).(Example 5)

(6) Co-expression as in (3) above of Des-Val-alpha globin andDes-Val-beta globin. (Example 6)

(7) Co-expression as in (6) above, but with the Des-Val-beta globin genepreceeding the Des-Val-alpha globin gene within the operon. (Example 6)

(8) Co-expression as in (3) above of (Des-Val)-alpha-(Gly-Gly)-alphaglobin and beta-globin and intracellular assembly into a di-alpha Hgb.(Example 8)

(9) Co-expression as in (8) above of (Des-Val-alpha-(GlyGly)-alphaglobin and a mutant (Nagai, Arg-Nagai, or Kansas) of beta globin andintracellular assembly into a mutant di-alpha Hgb. (Example 9)

(10) Co-expression as in (3) above of Des-Val-alpha globin and a Des-Valbeta-globin with the Presbyterian mutation and intracellular assemblyinto a mutant Des-Val Hgb. (Example 11).

(11) Co-expression as in (8) above of a (Des-Val)-alpha-(GlyGly)-alphaglobin and of a betal globin with the Presbyterian mutation. (Example11).

(12) Expression of di-alpha globin and beta-globin from separatepromoters on the same plasmid (Example 12).

(13) Co-expression of di-alpha globin and two copies of beta globin fromsame operon (Example 13).

(14) Expression of Di-beta Hgb (Example 14).

(15) Devised Hypothetical Protocol for co-expression of alpha globin andbeta-globin under control of separate promoters on the same plasmid(Example 16).

(16) Devised Hypothetical Protocol for co-expression of alpha andbeta-globin under control of different promoters on different plasmids(Example 17).

(17) Co-expression of α and β globin in S. cerevisiae (Example 19).

(18) Co-expression of di-α globin and β-globin in S. cerevisiae.(Example 23).

(19) Construction of Des-Val-alpha globin and Des-Val-beta globin in E.coli under lambda P_(L) control.

(20) Co-Expression of di-alpha globin and Des-Val-beta globin in E. coliunder lambda P_(L) control.

(21) Co-Expression of alpha and beta globin from separate plasmids indiploid strains of S. cerevisiae.

(22) Co-Expression of di-alpha globin and beta globin Presbyterianmutant in S. cerevisiae.

(23) Preparation of vectors for expression of di-alpha globins with-Gly- or -Pro-linkers.

(24) Evaluation of different strains and induction temperatures forexpression of di-alpha Hgb in E. coli.

(25) Co-Expression of alpha and mutant beta globin in S. cerevisiae,assembling to form low-affinity Hgb mutants.

(26) Construction of vectors for expression of monocysteine (asymmetric)mutants of di-alpha and di-beta hemoglobin.

(27) Hypothetical protocol for expression of a disulfide-bondedpseudooctamer.

An unexpected and surprising change in oxygen binding characteristics ofhemoglobin was observed upon replacement of the N-terminal valine withmethionine. As illustrated in Example 11, hemoglobin A₀ purified fromblood has a P₅₀ value of 4.3 with N=2.8 when measured at 25° C.DesFX-hgb produced in E. coli, a hemoglobin identical to A₀ except forthe addition of a methionine at the N-termini of the alpha and betachains, has essentially the same P₅₀ and N values. (Within experimentalerror, Example 7). Thus, the addition of a methionine, without alteringthe adjacent valine residue, has little or no effect on oxygen binding.On the other hand, a higher P₅₀ value, 6.6, was observed for desval-hgbproduced in E. coli, a hemoglobin in which the normal N-terminal valineof each chain was replaced with methionine. Cooperativity, as measuredby N, was virtually the same, however, for all three molecules.

A similar comparison was made for two hemoglobins each containing thePresbyterian mutation, one produced in E. coli (Example 11) and one inyeast. The E. coli hemoglobin was constructed with a Des-Val alphachain, i.e., the N-terminus had the normal valine replaced withmethionine. Oxygen binding was characterized by P₅₀ =19.8, N=2.5 at 25°C. and by P₅₀ =34.5 and N=2.5 at 37° C. (Example 11). The correspondingyeast coding region begins with an additional methionine codon in frontof the normal valine codon. Because this initial methionine is removedpost translationally in vivo, the purified hemoglobin has a normalN-terminal valine. For this molecule, P₅₀ =23 to 25 and N=2.5 whenmeasured at 37° C. Thus, in the above instances, the replacement of anN-terminal valine with an N-terminal methionine increased the P₅₀ value.Under physiological conditions, it is expected that the geneticallyfused Presbyterian hemoglobin produced in E. coli will deliver 20-30%more oxygen than the similar hemoglobin, with its altered N-terminus,produced in yeast.

A very large number of different plasmids are referred to in theExamples which follow. In order to highlight the relationships amongthese plasmids, a Table of vectors has been compiled (See Table 200).

EXAMPLE 1 Construction of FX-alpha Globin (pDLII-62m) and BetaGlobin(pDLII-10a) Expression Vectors

1.1 Materials and Methods

Unless otherwise stated all electroelutions, phenol/chloroformextractions, ethanol (EtOH) precipitations, alkaline-SDS plasmidpurifications, agarose electrophoresis and DNA manipulations werecarried out essentially as described by Maniatis et al. ("MolecularCloning" Cold Spring Harbor, N.Y., 1982).

The following abbreviations and definitions are used:ethylenediaminetetraacetic acid (EDTA); sodium dodecylsulfate (SDS);polyacrylamide gel electrophoresis (PAGE); dimethylsulfoxide (DMSO);dithiothreitol (DTT); isopropyl-beta-D-thiogalactopyranoside (IPTG);2×YT medium (16 g bacto tryptone, 10 g Bacto yeast extract, 5 g NaCl perliter water); SDS-PAGE loading buffer (0.125M Tris-HCl, pH 6.8, 20% v/vglycerol, 2% SDS, 2% 2-mercaptoethanol, 0.01% bromphenol blue);phosphate buffered TB medium (24 g yeast extract, 12 g tryptone, 4 mLglycerol, 17 mL 1M KH₂ PO₄, 72 mL 1M K₂ HPO₄ per liter water); Tris-EDTAbuffer (TE: 10 mM Tris, pH 8.0, 1 mM EDTA); Tris-borate, EDTA buffer(TBE: 0.089M Tris, 0.089M boric acid, 0.002M EDTA); Tris-acetate EDTAbuffer (TAE: 0.04M Tris, 0.04M acetic acid, 0.001M EDTA).

Protein electrophoresis was performed by the method of Laemmli, U. K.(Nature 1970, 227, 680-685) on 15% SDS-polyacrylamide gels.

E. coli JM109 cells were made competent as follows: Two hundredmilliliters of 2×YT medium was inoculated with 2 mL of an E. coli JM109overnight culture. The 200 mL culture was then incubated with shaking at37° C. for 1 hour. Cell were harvested by centrifugation at 6000 rpm at4° C. for 4 min in a Beckman JS13.1 swinging bucket rotor (BeckmanInstruments, Inc., Palo Alto, Calif.). The cells were resuspended in 50mL total of a buffer containing 45 mM MnCl2, 60 mM CaCl₂, 40 mM KOAc, pH6.2, 15% sucrose (w/v) 1.3% RbCl (w/v), and 7.5% (v/v) glycerol.Following centrifugation as above, the cells were resuspended in 20 mLof the same buffer and incubated at 0° C. for 30 minutes. Cells weredispensed in one milliliter aliquots and stored at -80° C. until used.

Unless otherwise stated, all restriction enzyme digests were performedunder conditions suggested by the manufacturer. DNA concentrations weredetermined by Beer's law using measured absorbance at 260 nm (A₂₆₀)against a water reference standard. For synthetic oligonucleotides thefollowing extinctions were used: E²⁶⁰ =0.05 mL/ug/cm and for doublestranded DNA E²⁶⁰ =0.02 mL/ug/cm.

1.2 Globin Gene Synthesis

Each globin gene was constructed from 14 separate oligonucleotidesranging in length from 50-85 base pairs. The FX-alpha globin gene wassynthesized from oligonucleotides SJH I-33a-f, SJH I-34a-f and SJHI-35a,b; FX-beta globin gene was synthesized from SJH I-36a-f, SJHI-37a-f and SJH I-38a,b. Each globin gene is preceeded by a short loadergene as previously described. Oligonucleotides were synthesized on aBiosearch 8600 instrument using beta-cyanoethylphosphoramidite chemistryon 1,000 angstrom CPG columns (0.2 mmole) (Beaucage, S. L. andCaruthers, M. H. Tet. Lett. 1981, 22, 1859-1862). The sequence of theseoligonucleotides is given in FIG. 4.

Unless otherwise stated, the oligonucleotides were cleaved from thecolumns using the following protocol: Approximately 0.5 mL of fresh,concentrated NH₄ OH was drawn into the column with a 1 mL syringe. TheNH₄ OH was allowed to react for 20 min then expressed into a glass vial(approximately 4 mL capacity). This process was repeated 2 times, thevial was filled to greater than 75% capacity with NH₄ OH, and heated at55° C., overnight. The samples were lyophilized, resuspended in 0.1 mLH₂ O and the concentration estimated by measuring A₂₆₀.

Two hundred micrograms of the individual oligonucleotides were purifiedby urea polyacrylamide gel electrophoresis. To do this, an equal volumeof 2× loading buffer (90% formamide (v/v), 0.5×TBE, 0.05% (w/v)bromophenol blue, 0.05% (w/v) xylene cyanol) was added to theoligonucleotide. The sample was heated at 95° C. for 10 min. and appliedto a 10% acrylamide gel containing 7M urea and 1×TBE. Electrophoresiswas at 800 volts for approximately 2 hrs. The full lengtholigonucleotide was visualized under ultraviolet light. That region ofthe gel was then excised and incubated in 3 mL of 100 mM Tris, pH 7.8,500 mM NaCl, 5 mM EDTA buffer at 60° C., overnight.

The oligonucleotide solution was further purified by reverse phasechromatography as follows: A C18 Sep-Pak cartridge (Waters Associates)was washed with 10 mL 100% methanol followed by 10 mL of H₂ O. Theoligonucleotide solution was applied to the column, washed with 20 mL H₂O and eluted with 3×1 mL aliquots of 50 mM triethylammonium acetate, pH7.3/methanol (1:1). The purified oligonucleotide was lyophilized, washedwith 100% ethanol, dried, and resuspended in 0.1 mL H₂ O. Theconcentration was determined by A₂₆₀.

The synthetic FX-beta gene sequence (included in FIG. 5) was constructedas follows: 100 pmole of the following oligo nucleotides were kinased in3 separate reactions. Reaction 1 contained oligonucleotides SJH I-36b,c, d, e, and f. Reaction 2 contained SJH I-37a, b, c, and e. Reaction 3contained SJH I-37d, f, and SJH I-38a. After combining the appropriateoligonucleotides, the solutions were lyophilized to dryness andresuspended in 16 uL of H₂ O. Two uL of 10× kinase buffer (0.5MTris-HCl, pH 7.4, 0.1M MgCl₂), 0.5 uL of 100 mM DTT, and 1 uL of 1.0 mMATP were then added. The reaction was initiated by addition of 1 uL (2U) of T4 polynucleotide kinase (IBI, Inc., New Haven, Conn.). Afterincubation at 37° C. for 1 hour, the reactions were heated to 95° C. for10 minutes to inactivate the kinase. The three reactions were combinedand 100 pmoles of oligonucleotides SJH I-36a and SJH I-38b were added.After addition of 10 uL of 100 mM Tris, pH 7.8, 100 mM MgCl₂, theoligonucleotides were allowed to anneal by incubating at 65° C. for 30min, 37° C. for 30 min, and 15° C. for 1 hour. Annealed oligonucleotideswere ligated by addition of ATP (1 mM, final) and DTT (10 mM final) and4 uL (20 U) T4 DNA ligase (IBI, Inc., New Haven, Conn.) and incubationat 15° C. for 1 hour. Aliquots of this ligation mixture were then cloneddirectly into M13mp19 (see below).

Oligonucleotides for the construction of FX-alpha globin were similarlypurified, kinased, annealed, and ligated. Before ligation into M13mp19,the full length FX-alpha globin gene was purified by electrophoresisthrough 0.8% agarose in 1×TAE buffer and electroeluted into dialysistubing using 0.5× TBE as the electroelution buffer by the method ofManiatis et al. Eluted DNA was phenol extracted, EtOH precipitated, andresuspended in 20 uL TE buffer. Aliquots were used for cloning intoM13mp19.

1.3 Phage Vectors

For cloning, a 2, 5 or 10 fold molar excess of the individual FX-alphaand FX-beta globin gene sequences were combined with 200 ng of doublecut, gel purified M13mp19-RF (New England BioLabs, Inc., Beverly, Md.)and ligated overnight at 15° C. in 50 uL ligation buffer (IBI, Inc., NewHaven, Conn.) containing 2 U of T4 ligase. FX-Alpha globin was clonedinto the Xmal/PstI sites of M13mp19. FX-Beta globin was cloned into thePstI/HindIII sites of M13mp19.

E. coli JM109 was transformed with the M13mp19 ligation mixturecontaining the FX-alpha or FX-beta globin gene sequences using thefollowing transformation protocol: Nine microliters of DMSO was added to0.25 mL of competent E. coli JM109 and the cells were incubated on icefor 10 min. Aliquots of the FX-alpha or FX-beta globin ligationreactions were added and incubated on ice for 40 minutes and at 42° C.for 3 min. One hundred microliters of a JM109 overnight culture wasadded to each transformation mix followed by 60 μL of a solutioncontaining 50 μL of 2% (w/v) 5-bromo-4-chloro-3-indolylgalactopyranoside in dimethylformamide and 10 μL of 100 mmisopropylthiogalactoside. Molten B-top agar (10 g Bacto tryptone, 8 gNaCl, 6 g agar per liter), 2.5 mL was added and the mixture poured ontoa B-bottom agar plate (10 g Bacto tryptone, 8 g NaCl, 12 g of agar perliter). Following incubation overnight at 37° C. colorless plaques(i.e., clones containing inserted DNA) were removed from the platesusing sterile transfer pipettes and inoculated into 1 mL of a JM109overnight culture diluted 1:100 in 2×YT media.

Bacteriophage clones were grown at 37° C. for 6 to 8 hrs. andcentrifuged in a microcentrifuge for 5 minutes. The cell pellets wereprocessed for M13mp19 RF using the alkaline-SDS method and resuspendedin 20 μL of TE buffer containing 20 μg/mL DNAase free RNAase (Sigma, St.Louis, Mo.). Aliquots (1-3 μL) of the RF preparations were digested with10-20 units each of the appropriate restriction enzymes (see above) andanalyzed on 0.8% agarose electrophoresis gels (see above).

M13mp19 clones containing the correct size inserts were grown in largerquantities to obtain single stranded phage DNA for sequencing.Thirty-five milliliters of 2×YT medium was inoculated with 0.3 mL of aJM109 overnight culture. After growth for 1 hour at 37° C., the 35 mLculture was inoculated with 200 μL of the appropriate phage-containingsupernatant. The culture was incubated for 6 hours at 37° C. and theculture supernatant collected by centrifugation at 9000 rpm for 10 minin a JS13.1 rotor. The phage were precipitated from 31 mL of supernatantby addition of 5 mL 4M NaCl and 4 mL 40% (w/w) polyethylene glycol 6000and recentrifuged as above. The phage pellet was resuspended in 0.4 mLof TE, extracted with phenol/chloroform/isoamyl alcohol (50:49:1 (v/v))three times, chloroform/isoamyl alcohol (49:1 (v/v)) one time, andethanol precipitated. The DNA pellet was resuspended in 20 μL TE andquantitated spectrophotometrically by A₂₆₀. One microgram of phage DNAwas used per set of sequencing reactions.

Sequencing was by the dideoxy method of Sanger (Sanger, F. S. et al.Proc. Nat. Acad. Sci. USA 1977, 74, 5463-5467) with M13 -20 and M13 -40universal primers (New England BioLabs, Beverly, Mass.) and genespecific primers (for FX-alpha globin the following primers were used:alpha-1 5'-CGTATGTTCCTGTCTTT-3'; alpha-2 5'-ACAAACTGCGTGTTGAT-3'; forFX-beta globin the following primers were used: beta-15'-GCTGGTTGTTTACCCGT-3'; beta-2 5'-ACCCGGAAAACTTCCGTC-3').

1.4 Expression Vectors pDL II-62m and pDL II-10a

The appropriate sequences were excised from the M13mp19 vector andcloned into the pKK-223-3 (Pharmacia/LKB, Piscataway, N.J.)) expressionvector under control of the Tac promoter (Brosius, J. and Holy, A. Proc.Nat. Acad. Sci., USA 1984, 81, 6929-6933). A DNA sequence encoding alphaglobin was removed by cutting with EcoRI and PstI. The globin containingfragment was gel purified and ligated into EcoRI and PstI double cut,gel purified pKK-223-3 using methods described above. E. coli JM109cells were transformed with the ligation reaction containing the desiredFX-alpha globin sequence (pDL II-62m) (FIG. 1) and selected for bygrowth on 2×YT media containing ampicillin (100 μg/mL). Individualclones were screened for the presence of the desired insert (yieldingpDL II-62m) by alkaline-SDS purification of plasmids and restrictionanalysis with the enzymes EcoRI and PstI using the methods describedabove. Cloning of the FX-beta globin sequence into the pKK-223-3expression vector, yielding pDL II-10a (FIG. 1) was done analogouslyexcept that restriction analysis and cloning was done with PstI andHindIII.

EXAMPLE 2 Separate Expression of Synthetic FX-Alpha and FX-Beta Globin

To assess the expression of the individual FX-globin gene products, E.coli JM109 clones transformed with either pDL II-62m or pDL II-10a wereinoculated into 2 mL of TB media containing ampicillin (100 μg/mL). Theinoculum was grown at 37° C. for 3-4 hours, then divided into two 1 mLaliquots. One of the aliquots was induced by the addition of IPTG (1 mM,final) and grown for an additional 3-4 hours. The cells were collectedby centrifugation, resuspended in 0.5 mL SDS-PAGE loading buffer andheated at 85° C. for 10 minutes. The total cell protein mixture waselectrophoresed on 15% SDS-polyacrylamide gels using authentichemoglobin as a molecular weight standard.

EXAMPLE 3 Polycistronic Coexpression of FX-alpha and FX-beta Globin GeneProducts from the same Operon (PDLII-66a) And Conversion ofFX-Hemoglobin to Hemoglobin

To achieve coexpression of FX-alpha and FX-beta globins from a singlepolycistronic operon, the FX-beta globin sequence from pDL II-10a wasexcised with HindIII and PstI, gel purified, and ligated intoPstI/HindIII cut and gel purified pDL II-62m. Ligation andtransformation conditions were identical to those described above. Notethat each globin cistron was preceeded by an "introduction" cistron aspreviously described, so that the entire Tac promoter driven operon hadfour cistrons. Clones were individually examined for the presence ofboth FX-alpha and FX-beta globin genes by digestion of plasmids withEcoRI and separation of fragments by electrophoresis through 0.8%agarose. Plasmids containing both genes (pDL II-66a, FIG. 1) produced afragment of approximately 1.0 kb following EcoRI digestion.

Clones containing pDL II-66a were grown in 2 mL of TB media containingampicillin (100 μg/mL) for 4 hours at 37°. The culture was divided intotwo 1 mL aliquots, one of which was induced with 1 mM IPTG. Incubationwas continued for 4 hours. Total cell protein extracts for both theuninduced and induced clones were examined by SDS-PAGE electrophoresisto confirm the coexpression of both FX-alpha and FX-beta globin.

To determine if the coexpression of both gene products resulted in theformation of tetrameric FX-alpha globin₂ FX-beta globin₂ protein, thefollowing experiments were performed. Two liters of TB medium containingampicillin (100 μg/ml) was inoculated with 20 mL of an overnight cultureof an FX-alpha/FX-beta expressing E. coli clone and grown to an opticaldensity at 600 nm (OD₆₀₀) of 2.1 at 37° C. The culture was induced withIPTG (2.5 mM final concentration) and grown to an OD₆₀₀ of 3.5.

The cells (40 gm) were collected by centrifugation at 10,000×g andsuspended in 80 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 25% sucrose,1 mM EDTA). Ten milliliters of lysozyme solution (18 mg/ml in lysisbuffer) was added and the mixture incubated on ice for 30 min. MgCl₂,MnCl₂, and DNAse I (Sigma, St. Louis, Mo.) were added to finalconcentrations of 10 mM, 1 mM and 10 μg/mL, respectively. The cells wereincubated at room temperature for 1 hour and an equal volume of asolution containing 1% one percent deoxycholic acid, 1% Nonidet P-40, 20mM Tris-HCl pH 7.5, 2 mM EDTA was added to the lysate.

Particulate material was removed by centrifugation at 10,000×g for 10min. The supernatant (-200 mL) was bubbled with carbon monoxide for 5min and dialyzed overnight against 4 liters of 10 mM NaPO₄ buffer, pH6.0. The cell-free extract was clarified by centrifugation at 10,000×gfor 10 min. To the supernatant was added 20 g DE-52 (Whatman. U. K.).The pH of the suspension was adjusted to 7.0 and the ion exchange resinwas removed by centrifugation. The pH of the supernatant was readjustedto 6.0 and the Supernant was loaded onto a CM-cellulose column (2.5×15cm) equilibrated in 10 mM NaPO₄, pH 6.0 at 4° C. The column was washedwith two bed volumes of 10 mM NaPO₄, pH 6.0 followed by a lineargradient of 10 mM NaPO₄, pH 6.9 to 20 mM NaPO₄, pH 9.0 (400 mL totalvolume). Fractions 36 to 42 contained a red solution and were combined;an aliquot of this solution was scanned from 650 nm to 400 nm revealinga spectrum identical to that for carboxyhemoglobin (FIG. 6). An aliquotof the same peak was analyzed by SDS-PAGE electrophoresis withhemoglobin as molecular weight standard and was found to contain twoprotein bands of approximate MW 15,500 and 16,200. As expected, thesebands migrated at a slightly slower rate than authentic hemoglobin(alpha MW=15,100; beta MW=15,850) presumably due to the five amino acidextension of the Factor X recognition sequence. On this basis thematerial was designated FX-hemoglobin.

FX-Hemoglobin (2.0 mg) was digested at room temperature for 2 hours in 3mL of 20 mM HEPES, pH 7.4, 0.1M NaCl, 10 mM CaCl₂ containing 2 mg oftrypsin (Sigma, St. Louis, Mo., 10,000 units/mg). SDS-/PAGEelectrophoresis confirmed the conversion of FX-hemoglobin to materialthat comigrates with native hemoglobin.

EXAMPLE 3A Distribution of FX-Globin Products from E. coli Expression

One hundred milliliter cultures of E. coli clones expressing Fx-alphaglobin (plasmid pDL II-62m), Fx-beta globin (plasmid pDL II-10a) andFx-hemoglobin (plasmid pDL II-66a) were started with 1 ml inocula ofovernight cultures. After growth for 4 hours, protein expression wasinduced by addition of IPTG to 1 mM final concentration. Incubation wascontinued for an additional 3 hours. The cells were collected bycentrifugation, weighed, and lysed as described above except that DNAasetreatment was done for 30 minutes on ice. The samples were centrifugedat 5000×g, 10 min and the supernatants (representing the soluble proteinfraction) brought to 2 ml final volume with H₂ O and frozen at -80° C.The pellets (representing the insoluble protein or inclusion bodyfraction) were washed twice in 5 mL 0.5% Triton X-100, 1 mM EDTA,resuspended in 2 ml H₂ O and frozen at -80° C.

Analysis of protein distribution was accomplished by SDS-PAGE andWestern blotting. The primary antibody was rabbit anti-human hemoglobinIgG. The western blotting protocol was according to the manufacturer'srecommendations (Proto Blot Western Blot AP system, Promega Corp.,Madison, Wis.). Samples of soluble and insoluble protein representing 60μg of wet cell weight were analyzed from the FX-alpha and FX-betaexpressing clones. Due to the greater level of expression in theFX-hemoglobin clone, material representing only 15 μg of wet cell weightwas analyzed.

As seen in Table 11 the distribution of the proteins varied. Fx-alphaglobin was detectable only in the soluble fraction while FX-beta globinpartitioned between the insoluble and soluble fractions of the cell.FX-Hgb, which stabilizes each separate subunit, was found only in thesoluble fraction, and at a concentration at least 2.6 times that of theindividually expressed subunits. These results indicate that FX-betaglobin is totally soluble only when allowed to assemble with FX-alphaglobin.

EXAMPLE 4 Construction And Expression of Mutant FX-Alpha/FX-BetaTetrameric Hemoglobin Expression Vector

Hemoglobin Beth Israel

pDL II-10a was digested with restriction enzymes SacI and SpeI, gelpurified, and isolated by electro-elution. Oligonucleotidesincorporating the appropriate codon change for Hemoglobin Beth Israel(beta¹⁰² asn→ser) (FIG. 7) were synthesized as previously describedabove to bridge the SacI to SpeI restriction sites. Purification andquantitation of the individual oligonucleotides was as previouslydescribed. The complementary oligonucleotides were annealed by heatingto 95° C. for 10 min. followed by slow cooling to room temperature overa 2 hour period. An aliquot of the annealed mixture was then combinedwith the SacI/SpeI digested, gel purified pDL II-10a plasmid at molarratios similar to those used for the initial FX-alpha and FX-betacloning. T₄ DNA ligase (2 U) was added and the mixture incubated for 1hour at room temperature. E. coli JM109 was transformed with thisligation mixture as previously described. Individual clones wereisolated, and plasmids purified and sequenced using primer beta-1 (vidasupra). Plasmid sequencing was done with a Sequenase kit (United StatesBiochemical Corp., Cleveland, Ohio) following the protocol supplied bythe manufacturer. The appropriate mutated beta globin sequence was thenexcised with HindIII and PstI, gel purified, and cloned into pDL II-62mas described above.

Other hemoglobin mutants

The synthetic genes encoding Hemoglobin Cheverly (beta⁴⁵ phe→ser)Hemoglobin Providence/MSR (beta⁸² lys→asp) and Hemoglobin beta⁶⁷ val→ileand Hemoglobin Kansas (beta¹⁰² asn→thr) were prepared similarly exceptwith synthetic oligonucleotides spanning the SacII→BglII, SalI→SpeI,NcoI→KpnI and SacI→SpeI restriction sites respectively (FIG. 7).Synthesis of the mutant oligonucleotides, restriction enzyme digestion,gel purification, and ligation conditions were identical to those usedfor Hemoglobin Beth Israel. All mutations were first cloned into plasmidpDL II-10a, appropriate clones were sequenced, and the mutated betaglobin gene was subcloned into PstI and HindIII digested pDL II-66a.Plasmid sequencing was accomplished as described previously. E. colicells were transformed, cultured, and induced as previously described.FX-hemoglobin mutants were purified by the method of Example 3. oxygenbinding of purified hemoglobin mutants is shown in Table 9.

EXAMPLE 5 Production of Synthetic Met-Alpha/Met-Beta Hemoglobin

5.1 Construction of Des-FX Alpha and Des-FX Beta Globin Genes

We established that E. coli could produce tetrameric fusion-hemoglobin.Elimination of the DNA sequences coding for the Factor X_(a) substraterecognition site on the N-terminal end of each peptide should result inproduction of synthetic (FX free; "des-FX") hemoglobin containing theN-terminal methionine as its only extra residue. pDL II-62m was digestedwith EcoRI and PstI to excise the sequence containing the FX-alphaglobin gene. The FX-alpha globin gene was then gel purified. pGEM-1(Promega Corp.) was linearized with EcoRI and PstI, gel purified, andthe FX-alpha globin gene ligated into the plasmid as describedpreviously. Clones (pGEM-FX-A) containing the FX-alpha globin gene inpGEM-1 were identified by digestion of purified plasmids with EcoRI andPstI followed by agarose gel electrophoretic analysis. FX-Alpha pGEM wasdigested with NdeI and EagI to remove the Factor X_(a) coding sequence(FIG. 5). oligonucleotides containing the DNA sequence encoding nativealpha globin were synthesized with ends compatible to NdeI and EagI(FIG. 8) restriction sites. After synthesis, the oligoncleotides werepurified, annealed, and ligated as described above. The sequence ofpGEM-desFX-alpha (pDL II-83a) was confirmed by dideoxy-sequencing of theplasmid using the T₇ promoter primer (Promega Corp., Madison, Wis.). Aclone containing the correct sequence was then digested with EcoRI andPstI. The des-FX alpha globin gene was gel purified, cloned intoEcoRI/PstI digested, gel purified pKK-223-3 to generate pDL II-86c. E.coli strain JM109 was transformed with the ligation mixtures andexpression of des-FX alpha globin by individual clones was determined byanalysis of total cell protein extracts of induced culture inocula (seeabove). Des-FX alpha globin, in contrast to FX-alpha globin, co-migrateswith authentic alpha globin on SDS-PAGE.

The des-FX beta globin sequence was prepared in an analogous fashionusing the FX-beta globin gene excised from pDL II-10a with PstI andHindIII. This gel purified beta globin sequence was then ligated intopGEM-1 that had been digested with the same two enzymes. A pGEM-1plasmid containing the FX-beta globin gene was digested with NdeI andSacII, gel purified, and used for construction of the des-FX beta globingene. The oligonucleotides conferring the desired sequence (FIG. 20)were synthesized, purified, annealed, and ligated into NdeI, SacII cutpGEM FX-beta to form pGEM-des-FX beta (pDL III-6f) as described above.After confirming that the sequence was correct the des-FX beta gene wasremoved with PstI and HindIII and gel purified. Des-FX beta was thenligated into the des-FX alpha-containing plasmid pDL II-86c using thePstI/HindIII sites. Clones containing both des-FX alpha and beta globingenes (pDL III-13e) (FIG. 9) were confirmed by EcoRI digestion ofpurified plasmids (see above), and screened for expression by comparisonof IPTG induced and non-induced cultures. Des-FX hemoglobin co-migrateswith native hemoglobin on SDS-PAGE.

5.2 Characterization of Met-Hgb (Des-FX Hgb)

Recombinant methionyl-Hgb has different reactivities than Hgb Ao in thepresence of chloride and phosphate ions (Table 10) and with changes inhydrogen ion concentration (FIG. 10). The reason for this is thought tobe the additional amino acid, methionine, on the N-termini of bothglobins. The N-terminal amino group of the alpha chain is important forthe change in P₅₀ by phosphate ion. Displacement of the N-terminal aminogroup in space or changing its electronic state by the addition of themethionine can alter these effects.

The increase in P₅₀ seen in the presence of inositol hexaphosphate ionis irrelevant for any Hgb found in solution because the concentration ofmonophosphate ion found in plasma is not enough to significantlyincrease the P₅₀. There is no inositol hexaphosphate ion found inplasma. The increase in P₅₀ needed for Hgb in solution to effectivelyoff-load oxygen can be best attained by incorporating mutations to makeHgb lower in affinity for oxygen.

The magnitude of change in P₅₀ with respect to chloride ion is notimportant physiologically. The effect, which is interesting in terms ofits biochemical mechanism, does not add a significant amount of oxygenoff-loading capacity to Hgb in solution. Some animal Hgb have extremelysmall chloride effects. Again, the chloride effect is thought to betransmitted by the alpha globin N-terminal amino group; withmethionyl-Hgb that effect could be changed because of the stearic changeor because the pKa of the methionine amino group is different than thatof valine.

The P₅₀ of Hgb is normally most dramatically changed by hydrogen ionconcentration. The so-called "Bohr effect" is also thought to, in part,involve the N-terminal amino group of the alpha globin. Numerous mutanthuman Hgb molecules have been shown to have altered Bohr effect changeswithout any physiological deficiency. There is an advantage in having alimited Bohr effect, as well as phosphate and chloride effects, in a Hgbmolecule to be used in solution for a number of different medical andbiochemical applications. In terms of the Bohr effect, there are severalapplications for methionyl-Hgb where it could be used in the morealkaline pH range, e.g., tissue culture, organ perfusion. FIG. 10depicts a preliminary experiment which indicated that the P₅₀ isactually greater for methionyl-Hgb than for Hgb Ao at greater than pH7.8. It will be noted from FIG. 10 that the slope of the P₅₀ to pH plotfor Met-Hgb is shallower than that for Hgb A₀, i.e., the Bohr effect issmaller. Subsequent experiments suggest that the difference in Bohreffect between Met-Hgb and Hgb A₀ is smaller than that shown in FIG. 10.

The main advantage for using a Hgb molecule with fixed changes in P₅₀relative to pH, chloride and phosphate, is that the practitioner willknow the oxygen off-loading capacity of the formulation without regardfor the specific conditions of its use.

EXAMPLE 6 Synthesis of Synthetic Hemoglobin (Des-Val Hgb) of Native Size

6.1 Construction of Des-Val-Alpha (pDL III-1a) and Des-Val-Beta (pDLII-95a) Globin Genes

DNA sequences encoding the globin genes in which the N-terminal valinecodon in each gene is replaced by an ATG (methionine) codon wereconstructed in a manner analogous to the des-FX clones except that theoligonucleotides inserted (FIG. 8) were ones encoding the amino acidsequences "met-leu . . . " and "met-his . . . " for alpha and betaglobin genes, respectively. Following confirmation of correct sequencefor both des-val alpha (pDL II-91f) and des-val beta (pDL II-95a) genesin pGEM-1, the des-val alpha globin gene was cloned into the EcoRI/PstIcut pKK-223 (see above) to create pDL III-1a. The des-val beta globingene from pDL II-95a was then cloned into pDL III-1a using thePstI/HindIII restriction sites.

More specifically, the des-val alpha transfer vector was prepared fromplasmid pDL II-62m as follows. Plasmid pDL II-62m was digested withEcoRI and PstI to excise the fragment containing the FX alpha globingene. The FX alpha globin gene was then gel purified. The plasmid pGEM-1(Promega Corp.) was linearized with EcoRI and PstI, gel purified, andthe FX alpha globin gene ligated into the plasmid as describedpreviously. It was necessary to subclone into pGEM-1 because redundantrestriction sites in pKK 223-3 prohibited the removal of the FX codingsequence directly from the individual FX-alpha and FX-beta globin genes.Clones containing the FX alpha globin gene in pGEM-1 were identified bydigestion of purified plasmids with EcoRI and PstI followed by agarosegel electrophoretic analysis. FX alpha pGEM-1 was digested with NdeI andEagI to remove the FX alpha coding sequence and oligonucleotidescontaining DNA sequences coding for native alpha globin in which theN-terminal valine is replaced by a methionine were synthesized with endscompatible to NdeI and EagI restriction sites. After synthesis,oligonucleotides were purified, annealed and ligated as described above.The sequence of pGEM des-val alpha (plasmid pDL II-91f) was confirmed bydideodoxy sequencing of the plasmid using the t7 primer (promega corp.,Madison, Wis.). A clone containing the correct sequence for des-valalpha (pDL II-91f) was then digested with EcoRI and PstI. The des-valalpha globin gene was gel purified, and ligated into EcoRI/PstIdigested, gel purified, pKK-223-3 to generate plasmid pDL III-1a.

The des-val beta globin transfer vector was prepared in an analogousfashion using the FX beta globin gene of plasmid pDL II-10a. FX-beta wasexcised from pDL II-10a with PstI and HindI. This gel purified betaglobin sequence was then ligated into pGEM-1 that had been digested withthe same two enzymes. A pGEM clone containing the FX beta globin genewas digested with NdeI and SacII, gel purified, and used forconstruction of the des-val beta globin gene. The oligonucleotidesencoding the desired sequence for des-val beta were synthesized,purified, annealed, and ligated into NdeI, SacII cut, pGEM FX-beta toform pGEM des-val beta (plasmid pDL II-95a). After confirming that thesequence was correct for des-val beta globin, the gene was removed withPstI and HindIII and gel purified.

6.2 Preparation of pDL III-14c and III-38b (des-Val-alpha/des Val betaPolycistronic Gene Clones)

Plasmid PDL III-1a containing the Des-Val alpha globin gene was digestedwith PstI and HindIII and gel purified. The Des-Val beta globin gene wasremoved from pDL II-95a using the same method. Following ligation andtransformation, individual clones containing the Des-Val alpha/des-valbeta globin coexpressing plasmid pDL III-14c were analyzed for Des-Valhgb production by IPTG induction and SDS-PAGE.

Plasmid pDL III-38b which contains the Des-Val beta globin gene 5' tothe Des-Val alpha globin gene was then constructed and analyzed.

Plasmid pDL III-1a containing the des-val alpha globin gene waslinearized with the restriction enzyme SmaI. The plasmid was thentreated with bacterial alkaline phosphatase to remove the 5'-phosphategroups, phenol extracted, ethanol precipitated, and resuspended in TEbuffer as above. A pGEM-1 clone (pDL II-95a) containing the des-val betagene was digested with HindIII, phenol extracted, ethanol precipitatedand resuspended in a ligation mixture containing a 50:1 molar ratio of aHindIII-SmaI linker to plasmid. This ligation mixture was then digestedwith SmaI and the beta globin fragment now containing SmaI restrictionsites on both the 5'- and 3'-ends was gel purified and added to aligation reaction containing the linearized form of plasmid pDL III-1a,above, thus obtaining plasmid pDL III-38b. The orientation of the alphaand beta globin genes in pDL III-14c and pDL III-38b was confirmed byrestriction analysis.

Cells of E. coli strain JM109 were transformed with pDL III-14c or pDLIII-38b and grown in 2×YT media containing ampicillin. Colonies wereinduced with IPTG as above. Individual clones were analyzed for theirability to produce des-val alpha and des-val beta globin polypeptides bySDS-PAGE and Western blotting. There was no appreciable differencebetween expression of immunoreactive des-val alpha or des-val betaglobins from the alpha→beta orientation (pDL III-14c) or the beta→alphaorientation (pDL III-38b).

EXAMPLE 7 Analysis and comparison of the Functional Characteristics ofRecombinant Des-FX and Des-Val Hemoglobins

JM109 cells expressing either des-FX hemoglobin (dFX-hgb) or des-Valhemoglobin (dV-hgb) were grown to an OD600 of 15 in a 10 liter fermenterand then induced by the addition of 300 μm IPTG. Induction period wasfor 6 hrs. Cells were harvested by centrifugation and frozen at -80° C.until processed.

For purification of hgb approximately 200 g of cells were resuspended in350 mL of 50 mM sodium phosphate (NaPi) buffer, pH 7.0 containing 200units of aprotinin/mL and 20 μg/ml DNAase 1. Cells were then lysed bythe addition of 1 mg/mL of lysozyme and 4 passages through a Dynomill.Cellular debris was removed by centrifugation at 10,000 rpm in a BeckmanJA14 rotor at 4° C. for 40 min. The supernatant was added to 200 mL of ahemoglobin-binding resin equilibrated with 10 mM NaPi, pH 7.0. The pHwas adjusted to 7.0 with 10M NaOH or concentrated phosphoric acid. Theresin was then loaded into a 5×30 cm chromatography column and allowedto settled. The column was then washed with 2.5 column volumes of 10 mMNaPi. pH 7.0 containing 100 units/mL aprotinin. Hgb was eluted from thecolumn in 20 mM Tris-HCl, pH 7.5 containing 100 units/mL aprotinin. Thispartially purified hgb was then 0.2 micron filtered and loaded onto a1.6×10 cm Mono-Q anion exchange column equilibrated with 20 mM Tris-HCl,pH 8.0. Hgb was eluted using a linear gradient of 0 to 0.4M NaCl in 20mM Tris-HCl, pH 8.0. The material was then loaded onto a 1.6×10 cmMono-S cation exchange column. Hemoglobin was eluted with a lineargradient of 10 mM NaPi, pH 7.0 to 10 mM NaPi, pH 8.5, 160 mM NaCl. Themajor peak of hgb was collected, concentrated to approximately 100mg/mL. and used for analysis.

Functionality of the recombinant hgb was evaluated using a Hemoxanalyzer at 25° C. in 50 mM HEPES, pH 7.4 containing 0.1M Cl⁻. Thefollowing oxygen binding data were obtained:

    ______________________________________                                        SAMPLE            P.sub.50                                                                            N                                                     ______________________________________                                        Ao                4.3   2.9                                                   dFX-hgb           3.3   2.6                                                   dV-hgb            6.6   2.7                                                   ______________________________________                                    

Of significance in these data are the following:

1) The addition of an extra amino acid, methionine, on the N-termini ofalpha and beta globins (dFX-hgb) appears to reduce slightly the P₅₀ ofthe molecule but has little effect upon cooperativity (N).

2) Replacement of the N-terminal valines of alpha and beta globins withmethionine (dV-hgb) increases the P₅₀ of the molecule but has littleeffect upon the cooperativity (N).

EXAMPLE 8 Polycistronic Co-expression of Des-Val-Alpha/Alpha (Di-Alpha)and Des-Val Beta Globin

The overall synthetic plan for the preparation of a plasmid (pDLIII-47a) which co-expresses di-alpha globin and beta globin is givenbelow. The starting materials are the commercially available transfervectors M13mp19-RF, pKK 223-3 and pGEM-1, and the syntheticoligonucleotides described in the examples.

For convenience, our manipulations began with plasmids, pDL II-62m andpDL II-10a. Plasmid pDL II-62m was obtained by cloning the "FX alphaglobin" gene into pKK 223-3 downstream of the Tac promoter. Plasmid pDLII-10a was prepared by an analogous insertion of the "FX beta-globin"gene.

As set forth in greater detail in FIG. 12, the "FX alpha globin" operonencodes two cistrons, the first expressing an octapeptide "leader", andthe second, alpha globin preceded by Met-Ile-Glu-Gly-Arg. The latterfour amino acids constitute a recognition site for Factor X cleavageactivity. The "FX beta globin" operon is similarly constructed.

Since the Factor X recognition site was not needed here, the geneticmaterial was manipulated to excise the "FX" codons. (This could havebeen avoided by synthesizing the desired di-alpha globin anddes-val-beta globin genes directly rather than using the FX-alpha andFX-beta genes of pDL II-62m and pDLII-10a.) The FX-alpha globin genecassette was excised and cloned into pGEM-1 to obtain pGEM FX-alpha(pGEM FX-A). Similarly, the FX-beta gene of pDL II-10a was transferredto pGEM-1 to obtain pGEM FX-beta (pGEM FX-B).

The recognition site (FX)-encoding sequence could now be removed frompGEM FX-alpha and pGEM FX-beta to obtain pDL II-91f and pDL II-95a,respectively. The des-val alpha globin gene of pDL II-91f was reclonedinto pKK 223-3 to generate pDL III-1a, the gene being operably linked tothe Tac promoter of pKK-223-3. The des-val beta globin gene of pDLII-95a was purified and inserted downstream of the des-val alpha globingene of pDL III-1a to form a single transcriptional unit which wouldencode a polycistronic alpha globin/beta globin mRNA, see pDL III-14c.Finally, a synthetic oligonucleotide comprising the desired di-alphalinker encoding sequence and another copy of the alpha globin gene wasinserted into pDL III-14c to create pDL III-47a, wherein a Tac promotercontrols transcription of a di-alpha globin gene and a des-val betaglobin gene.

8.1 Preparation of pDL III-47a (di alpha/beta globin clone)

The EagI and PstI restriction fragment containing most of the alphaglobin gene from the plasmid pDL II-91f was gel purified and ligated toa synthetic DNA linker containing the sequence from the BstBI site ofthe alpha globin gene to the codon (wild-type Arginine) for its carboxylterminus, a variable glycine-encoding linker (for example, FIG. 12,RGGV, a di-glycine followed by α Val; other possibilities include RGM,RGV, RGGV, etc., See Table 200), and the codons for the amino terminalregion of alpha globin to the EagI site (FIG. 12). After digesting thisligation mixture with Pst I, the resulting fragment was cloned intoBstBI/PstI-cut pDL III-14C to create plasmid pDL III-47a (RGM-di-alpha).Plasmids pDL III-82a (RGGV-di-alpha), pDL IV-8a (RGV-di-alpha), pDLIV-976 (RV-di-alpha) and pDL IV-66a (RGGGV-di-alpha) were similarlyconstructed to incorporate the indicated changes in the di-alpha codingsequences.

8.2 Expression of Di-alpha/Beta Hemoglobin

Individual E. coli clones were analyzed by Western blotting forproduction of dimeric alpha globin protein in combination with monomericbeta globin. Appropriate plasmid construction was confirmed by digestionwith EcoRI and 0.8% agarose gel electrophoresis. The EcoRI fragmentpresent in the di-alpha constructs is approximately 1450 bp.

Expression of genetically fused hemoglobin was accomplished using theIPTG induction protocol and S-sepharose purification of recombinanthemoglobin. E. coli cells (400 ml) were grown to an OD₆₀₀ of 3.0 andinduced with 1 mM IPTG. The cells were allowed to continue to grow foranother 4 hours and then harvested by centrifugation. The cell pelletwas resuspended in the 10 mM sodium phosphate, pH 6.0 containing 1 mMbenzamidine, 1 mM EDTA and 0.1% Triton-X100. The cell suspension wasthen sonicated, centrifuged at 15,000×g for 15 minutes, and thesupernatant loaded on to an S-Sepharose column equilibrated with 10 mMsodium phosphate pH 6.0. After the sample was loaded on the column, thecolumn was washed with 10 bed volumes of 10 mm sodium phosphate pH 6.8.The dialpha hemoglobin was eluted from the column with 10 mM sodiumphosphate, pH 7.4, 30 mM NaCl. Confirmation of hemoglobin production wasaccomplished by visible light spectroscopy, SDS-PAGE, and Western blotanalysis of purified material.

Oxygen binding measurements were made at 37° and 25° C. in a HemoxAnalyzer (Southampton, Pa.). The solutions were 50 mM HEPES, pH 7.4,0.1M NaCl. The measured P50's and Hill coefficients are given below:

    ______________________________________                                                     P50                TEMP                                          Protein      (torr)      N      (°C.)                                  ______________________________________                                        RV-di-alpha  2.2         1.73   25                                                         5.0         1.49   37                                            RGV-di-alpha 4.5         2.04   25                                                         9.9         2.07   37                                            RGGV-di-alpha                                                                              8.2         2.39   25                                                         15.0        2.22   37                                            RGGGV-di-alpha                                                                             7.1         2.57   25                                            ______________________________________                                    

EXAMPLE 9 Preparation of Di-Alpha Hemoglobin Low Affinity Mutants

In order to reduce the oxygen affinity of recombinant di-alphahemoglobin, several mutations were introduced into the beta globinpolypeptides using synthetic oligonucleotides. The restriction sitesused to incorporate these mutants are shown in Table 3. For insertion ofthe Nagai (beta Val 67→Ile) and Arg-Nagai (also beta Lys 82→Arg)mutations, the des-Val-beta plasmid pDL II-95a was digested with therestriction enzymes NcoI and KpnI and gel purified. Oligonucelotidesspanning these two restriction sites and containing the appropriatecodon changes were synthesized, purified, annealed and ligated into thegel purified plasmid. Following confirmation of correct sequence, themutant des-Val-beta globin gene was excised with PstI, HindIII, gelpurified, and cloned into plasmid pDL III-47a. The beta globin genecontaining the Kansas mutation (beta Asn 102=>Thr) was similarlyconstructed using SacI and SpeI restriction sites. Mutated codons forall of these beta globin mutations are shown in lower case letters inTable 3.

EXAMPLE 10 Characterization of Di-Alpha Hemoglobins

Oxygen Binding

Oxygen binding measurements were made at 37° C. in a Hemox Analyzer(Southampton, Pa.). The solutions were 50 mM Bis-Tris, pH 7.4, 0.1M NaCland 60 uM heme equivalents of di-alpha hemoglobin. The solutions weremeasured between 120 and 1.5 torr oxygen pressure. P₅₀ values are givenin Table 4.

In Vivo Half Life

Di-Alpha Hgb (wild type) containing a gly-gly linker between alpha₁ andalpha₂ (RGGV-di-alpha, pDL III-82a) was prepared as describedpreviously. The protein was formulated in 20 mM NaPO₄, pH 7.4 at aconcentration of 95 mg/ml. Di-alpha Hgb was infused into maleSprague-Dawley rats 388-426 gm) at a dose of 875 mg/kg through a centralvenous catheter over 20-30 sec. Samples of blood were drawn at 2, 30,80, 90, 120, 150, 180, 210, and 240 min. into heparinized vials. Theblood was centrifuged to remove red blood cells and the plasmahemoglobin was assayed by absorbance at 540 nm. The percent hemoglobinremaining versus time was determined by comparison to the 2 min timepoint which was assumed to be a homogeneously mixed sample. The sameexperiment was repeated with non-crosslinked des-val Hgb at aconcentration of 100 mg/ml. The data were averaged for each sample andplotted as percent Hgb remaining against time after infusion. Themeasured half-lives were 205 min and 104 min respectively for di-alphaHgb and des-val Hgb, respectively.

EXAMPLE 11 Co-expression of Wild Type Des-Val Alpha Globin and Di-AlphaGlobin with Des-Val Beta Globin Containing the Presbyterian Mutation

Plasmid pSGE1.0-E4 is shown in FIG. 34 and contained the followingmodifications as compared to other expression vectors derived fromplasmid pKK223-3:

1) The plasmid now contains a functional tetracycline resistance gene.

2) The lacI gene which encodes for the lac repressor protein has beenincorporated into the plasmid. The lac repressor protein represses theTAC promoter until induction with IPTG. The repressor gene was insertedinto the plasmid to permit transformation of E. coli cell lines which donot have endogenous lac repressor genes.

The desVal beta globin gene in pSGE1.0E4 containing the Presbyterianmutation had been constructed by insertion of a complementary pair ofsynthetic oligonucleotides between the Sacl and Spel restriction sitesof pDL II-95a.

The following oligonucleotides (Pres-A and Pres-B) were used toconstruct the Presbyterian mutation in dVal-beta globin.

    __________________________________________________________________________    Pres-A                                                                             5'    CCACTGCGACAAACTGCACGTTGACCCGG (Continued below)                    Pres-B                                                                             3'TCGAGGTGACGCTGTTTGACGTGCAACTGGGCC (Continued below)                         .sup.    SacI                                                            Pres-A                                                                             AAAACTTCCGTCTGCTGGGTaaaGTA                                                                         3'                                                  Pres-B                                                                             TTTTGAAGGCAGACGACCCAtttCATGATC                                                                     5'                                                  SpeI                                                                          __________________________________________________________________________

Following digestion with the two restriction enzymes pDLII-95a was gelpurified to remove the wild type encoding DNA fragment. The annealedoligonucleotides were then ligated into the plasmid to create plasmidpJRV-83a. The HindIII to PstI fragment from this plasmid was thenligated into pSGE0.0E5(Table 200, #45a ) to create pSGE1.0E5(Table 200,#46). The BamHI fragment encoding the tet R 5' end was then ligated intopSGE1.0E5 to create pSGE1.0E4. Following transformation of JM109 cells,individual colonies were selected and analyzed for production of alphaand beta globins by IPTG induction and SDS-PAGE. Dideoxynucleotidesequencing was used to confirm the presence of the Presbyterianmutation.

The mutant hemoglobin was produced, purified, and analyzed as describedin Example 7 above. The oxygen affinity (P50) and cooperativity (N) ofwild-type Hgb A₀ (purified from blood), dV-Hgb expressed from pSGE0.0E4(FIG. 14), and dV-Hgb containing the Presbyterian mutations expressedfrom pSGE1.0E4 (FIG. 34), were compared:

    ______________________________________                                        SAMPLE            P.sub.50                                                                             N                                                    ______________________________________                                        Ao                4.3    2.9                                                  dv-hgb            6.6    2.7                                                  dv-hgbPres        19.8   2.5                                                  ______________________________________                                    

It should be noted that the Presbyterian mutation, which results in thechange of beta asparagine 108 to lysine, cause a large decrease in theaffinity of the molecule for oxygen but does not substantially affectthe cooperativity of the molecule.

The beta chain Presbyterian mutation has also been co-expressed withdi-alpha globin containing a single glycine linker utilizing plasmidpSGE1.1-E4 (FIG. 15). The result shown below indicates that the joiningof the carboxy terminus of alpha 1 to the amino terminus of alpha 2 hasrelatively little effect on oxygen binding and cooperativity.

    ______________________________________                                        SAMPLE            P.sub.50                                                                             N                                                    ______________________________________                                        dv-hgbpres        19.8   2.5                                                  dialpha/Pres      17.2   2.4                                                  ______________________________________                                    

EXAMPLE 12 Construction of a Two Promoter System for the Co-expressionof Di-Alpha Globin and Beta Globin

In this example, a di-alpha globin gene is operably linked to onepromoter and a beta globin gene to a second promoter, but both genesreside on the same vector. Compare Examples 16 and 17, infra.

Oligonucleotides (see below) encoding the sequence of the complementarystrands of the TAC promoter (syn pTAC) and appropriate restrictionenzyme sites were synthesized, gel purified, and annealed. Notice thatthe sequence complementary to the Xbal restriction enzyme site wasdesigned to eliminate this restriction site when ligated into anauthentic Xbal site. This was done to facilitate future manipulation ofsyn pTAC.

    __________________________________________________________________________    Sequence of syn PTAC                                                          __________________________________________________________________________     BamHI PstI                                                                   5'GATCCTGCAGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGG (Cont'd)                  3'    GACGTCTCGACAACTGTTAATTAGTAGCCGAGCATATTACACACC (Cont'd)                                   XbaI COMP                                                    .sup.    AATTGTGAGCGGATAACAATTTCACAC                                                                  3'                                                    .sup.    TTAACACTCGCCTATTGTTAAAGTGTGGATC                                                              5'                                                    __________________________________________________________________________

Plasmid pDL III-47a (FIG. 13) was digested with the restriction enzymesBamHl and Xbal, and the plasmid, now containing as an insert only partof the beta globin gene from the Xbal site to the HindIII site (FIG.13a), was gel purified.

Syn pTAC was then ligated into the plasmid to create plasmid pDL IV-64aand JM109 cells were transformed. Individual transformants were isolatedand analyzed by IPTG induction and SDS-PAGE to confirm the presence of afunctional TAC promoter.

Plasmid pDL III-47a was digested with the restriction enzymes Pstl andHindIII and gel purified to remove the beta globin coding sequences.Plasmid pDL IV-64a was digested with these same enzymes and the fragmentencoding syn pTAC/beta gel purified. Ligation of the syn pTAC/betafragment into the Pstl/HindIII digested pDL III-47a created plasmid pDLIV-67a (FIG. 16). Individual transformants were screened for productionof di-alpha and beta by IPTG induction and SDS-PAGE.

The dVal-beta globin gene under the control of syn pTAC was also ligatedinto another location in pDL III-47a. Syn pTAC/beta was removed from pDLIV-64a by digestion with Hind III and Pstl. The restriction siteoverhangs were filled with T4 polymerase and blunt end ligated into thePvu II site of PDL III-47a to create plasmid pJR VI-54a (FIG. 17). IPTGinduction and SDS-PAGE analysis were as previously described.

Results of induction experiments as evaluated by SDS-PAGE indicated thatcontrol of expression of di-alpha and beta by separate promoters gavelittle increase in the expression of either protein. Similarly,insertion of a second beta globin gene under regulation of a separatepromoter had little effect upon production of the proteins.

EXAMPLE 13 Insertion of a Second Translationally coupled Beta GlobinGene Into the Di-Alpha/Beta Expression Plasmid

The expression plasmid SGE1.1E4 (FIG. 15) contains a di-alpha globingene followed by a single beta globin gene. Translation of thepolycistronic mRNA results in the non-equimolar production of two alphaglobin proteins and one beta globin protein. Hemoglobin is composed ofequimolar quantities of alpha and beta globin, suggesting that thisexpression system may underproduce beta globin. In order to test thishypothesis, we inserted a second beta globin gene in front of thedi-alpha globin gene and analyzed expression of recombinant hemoglobinfrom this plasmid. Expression of all genes from a single promoter oughtto exert the greatest control over stoichiometry.

Construction of a di-alpha/beta expression plasmid which results inequimolar synthesis of alpha and beta globin proteins and synthesis ofgenetically stabilized hemoglobin which cannot dissociate intoalpha/beta dimers is novel. To our knowledge no such hemoglobin moleculehas ever been produced or shown to function as an oxygen carrier.

Plasmid pSGE1.1E4 (FIG. 15) was digested with the restriction enzymesHindIII and PstI. The beta globin gene was gel purified, T4 polymerasefilled, and ligated into SmaI linearized pSGE1.1E4. Following ligation,the mixture was again digested with SmaI to linearize plasmids notcontaining a second beta globin gene. The mixture was then transformedinto E. coli strain JM109. Plasmids from individual isolates werescreened for the presence of a second beta globin gene by digestion withthe restriction enzyme ScaI. An individual isolate was designatedSGE157. The plasmid was designated pSGE1.1.1E4 (FIG. 29).

A 2 liter fermentation was performed to obtain material for purificationand functionality determinations. Hemoglobin in crude cell extracts,quantitated by the difference assay, was 2.8 mg/gm cell paste. Thisassay allows us to measure the concentration of functional hemoglobin.The addition of a second beta gene appears to have no significant effecton the production of functional hemoglobin.

EXAMPLE 14 Construction of Di-Beta Globin Expression Vector andProduction of Di-Beta Hemoglobin

The general flowchart for this protocol is shown in FIG. 30.

a. Elimination of the NheI site from plasmid pGEM-1

Plasmid pGEM-1 was digested with restriction enzyme NheI, T4 polymerasefilled, ligated, redigested with NheI, and transformed into E. colistrain SGE127.

Strain SGE127 was obtained by screening JM109 for phage resistance.

Plasmids were isolated from individual colonies and screened for loss ofthe NheI site by digestion with NheI. An E. coli isolate containing theappropriate plasmid was grown up. The plasmid was purified anddesignated pSGE223.

b. Cloning of Beta Asn¹⁰⁸ →Lys into pSGE223

The gene for beta globin containing the Presbyterian mutation, ASN¹⁰⁸→Lys, was excised from plasmid pSGE1.1E4 (FIG. 15) with the restrictionenzymes PstI and HindIII and gel purified. Plasmid pSGE223 was similarlydigested and gel purified. The Beta fragment was ligated into pSGE223and transformed into SGE127. Individual isolates were screened for thepresence of the Beta globin fragment by PstI and HindIII digestion. AnE. coli isolate containing the appropriate plasmid was grown up. Theplasmid was purified and designated pSGE224.

c. Insertion of the di-beta linker sequences

Plasmid pSGE224 was digested with the restriction enzymes BamHI andSacII and gel purified. Annealed complementary syntheticoligonucleotides encoding the 2, 3, 4, or 5 glycine (GGT codon) linkersequences were ligated into the plasmid and transformed into SGE127.

Isolates were screened for the presence two NheI sites. E. coli isolatescontaining the appropriate plasmids were grown up. The plasmids werepurified and designated pSGE 229 (5 glycine linker), pSGE232 (4 glycinelinker), pSGE233 (3 glycine linker), or pSGE231 (2 glycine linker).

d. Construction of the di-beta globin gene containing the 5 glycinelinker

Plasmid pSGE 229 was digested with NheI. The fragment containing the 5glycine linker and the beta globin gene was gel purified and ligatedinto NheI digested plasmid pSGE224. Individual isolates were screened bydigestion with the restriction enzymes HindIII and PstI. An appropriateisolate was grown up. The plasmid was purified and designated pSGE 234.

e. Construction of pSGE 0.0E5

Plasmid pSGE0.0E4 was digested with BamHI to remove the 5' codingsequences for the tet resistance gene. After gel purification, theplasmid fragment was re-ligated and transformed into E. coli strainJM109. Isolates were selected for resistance to ampicillin andsensitivity to tetracycline. An individual isolate was grown up, theplasmid purified and digested with BamHI to confirm loss of the BamHIfragment. The isolate was designated SGE0.0E5. The plasmid wasdesignated pSGE0.0E5.

f. Construction of pSGE1.05E5

Plasmids pSGE234 and pSGE0.0E5 were digested with HindIII and PstI. Thedi-beta-containing fragment was gel purified from pSGE234 and ligatedinto gel purified pSGE0.0E5 from which the beta fragment had beenremoved. Following transformation, individual isolates were screened bySDS-PAGE for their ability to produce alpha and di-beta globin proteinssubsequent to induction with IPTG. An appropriate isolate was grown upand designated strain SDGE242. The plasmid was designated pSGE1.05E5.

g. Construction of pSGE1.05E4

The BamHI fragment containing the 5' end of the tetracycline resistancegene was gel purified from plasmid pSGE1.1E4 and ligated intoBamHI-linearized plasmid pSGE1.05E5. Isolates were selected by platingthe transformation mixtures on tetracycline containing nutrient agarplates. Individual isolates were screened for their ability to producealpha and di-beta proteins subsequent to IPTG induction. An individualisolate was grown up and designated SGE245. The plasmid was designatedpSGE 05E4 (FIG. 31).

Production of di-beta hemoglobin (5 glycine linker) was achieved withthis expression vector in E. coli but:

a. expression levels were low compared to our di-alpha/beta expressionsystem (4 to 5 fold lower yield).

b. the oxygen affinity of the alpha/di-beta hemoglobin was high P₅₀ =5.9torr (±0.6 torr) vs. .sup.˜ 32 torr for the di-alpha/beta molecule. (at37° C.) However, cooperativity, as measured by the Hill constant (n) wassimilar to the di-alpha construct (n=2.0±0.2 for both di-beta anddi-alpha).

Although a molecule with such a low P₅₀ may not be useable as bloodsubstitute or an oxygen-carrying plasma expander, it may have otheruses. For example, it may be particularly useful for delivering oxygento regions of tumors where oxygen tension is particularly low. Enhancedoxygen delivery during radiation or chemotherapy is known to enhance thetherapeutic effects of anti-neoplastic agents:

REFERENCES

(1) Dowling, S., J. J. fischer and S. Rockwell. (1991) A clinical studyof fluosol and hyperbaric oxygen as an adjunct to radiation therapy.Biomat. Art. Cells and Immobil. Biotech 19, 377.

(2) Herman, T. S. and B. A. Teicher (1991) Enhancement of radiationtherapy by an experimental concentrated perfluorooctylbromide (Oxygent)emulsion in the Lewis lung carcinoma. Biomat. Art. Cells and Immobil.Biotech. 19, 395.

(3) Holden, S. A., B. A. Teicher and T. S. Herman (1991) Effect of aPFOB Emulsion (oxygent) and carbogen breathing on the tumor cellsurvival of the FSaIIC fibrosarcoma after treatment with antitumoralkylating agents. Biomat. Art. Cells and Immobil. Biotech. 19, 399.

EXAMPLE 15 Hypothetical Protocol for Development of Linkers by Mutationand Selection

In this hypothetical example, the linker suitable for genetic fusion ofglobin subunits is obtained by mutagenesis of a linker-encoding DNAsequence and selection for functional linkers. Oligonucleotides spanningthe BstBI site of alpha₁ to the EaqI site alpha₂ will be synthesizedsuch that the six nucleotides comprising the preferred glycine-glycinelinker are randomized. By randomizing these nucleotides, codons for allcombinations of amino acids will be present in the oligonucleotidemixture. Following purification and annealing of the oligonucleotidesthey will be used to construct the di-alpha/beta co-expression genes asdescribed above.

Clones containing the various di-alpha/beta plasmids will then bescreened for production of increased levels of recombinant hemoglobinusing a protocol developed at the Company. E. coli clones will bearrayed on nitrocellulose filters overlayed on 2×YT-ampicillin platescontaining 1 mM IPTG. Following overnight incubation of 37° C., theplates will be sealed in a plastic bag in which the air has beendisplaced by carbon monoxide (CO). CO binding to intracellularrecombinant hemoglobin produces a distinctive red color in the E. colicolonies. Colonies producing the most intense red color will be furtheranalyzed.

In this experiment the assumption is made that certain combinations ofamino acids in the di-alpha linker will permit more stable folding ofthe individual, linked alpha globin chains and, therefore, result ingreater levels of production of intracellular recombinant hemoglobin.This increase level of production will result in a more intense redcolor in the appropriate E. coli clones.

After selection of several clones producing higher levels of recombinanthemoglobin, more detailed analyses will be done on individual clones todetermine the optimal di-amino acid linker. The analyses will includedetermination of quantities of recombinant hemoglobin produced, oxygenaffinity, and protein stability. Finally, clones found to be producingthe best quality recombinant hemoglobin will be DNA sequenced todetermine the amino acids comprising the linker.

This technique is equally applicable to other globin pseudodimers(di-beta and alpha/beta).

EXAMPLE 16 Hypothetical Protocol for the Synthesis of PlasmidsContaining alpha and beta globin genes under the regulation of twoseparate promoters on the same plasmid.

It is anticipated that recombinant hemoglobin can be expressed fromconstructs where the different globin genes are under the control ofseparate promoters. This situation would yield two separate mRNA's; onewith a dicistronic sequence encoding an alpha globin gene and anotherwith a dicistronic sequence encoding a beta globin gene. Forconstruction of an expression system in which both the alpha and betaglobin genes are under the regulation of separate promoters, on the sameplasmid, the following protocol would initially be used. Plasmid pDLIII-1a containing the des-val alpha globin gene would be digested withthe restriction enzyme BamHI, reacted with bacterial alkalinephosphatase, phenol extracted, ethanol precipitated, and resuspended inTE buffer. The plasmid pJR IV 50-a which is the pKK expression plasmidcontaining the des-val beta construct, would then be digested with therestriction enzymes BamHI and PvuI, to excise a fragment from theplasmid containing the P_(tac) promoter, the des-val beta sequence, thetranscriptional terminator sequence and a portion of the ampicillinresistance gene. Following gel purification of this fragment, aPvuI-BamHI linker would be synthesized and ligated onto the insert. Theinsert would then be back-cut with BamHI to generate BamHI compatiblesites on both the 5'- and 3'- ends of the insert. This insert would thenbe cloned into BamHI linearized plasmid pDL III-1a, resulting in aplasmid in which a translationally coupled des-val beta globin geneunder regulation of one P_(tac) promoter is positioned on the 3' side ofa translationally coupled des-alpha globin gene under regulation of aseparate P_(tac) promoter. Restriction enzyme mapping would be used toconfirm the orientation of the beta globin containing insert. E. coliJM-109 would be transformed with the plasmid containing the separateP_(tac) --globin constructs and grown in media containing ampicillin toisolate clones containing the plasmid. The clones containing the plasmidwould then be induced with IPTG and expression of des-val alpha globin,des-val beta globin and des-val hemoglobin would be assayed by SDS-PAGEanalysis, Western blotting with anti-hemoglobin antibodies and isolationof desval hemoglobin by standard chromatographic methods.

Alternatively, coexpression of both globin genes could be achieved fromDNA sequences on separate vectors under the control of separatepromotors.

EXAMPLE 17 Hypothetical Protocol for the Construction of VectorsContaining alpha and beta globin genes under the regulation of separatepromoters and on different vectors.

E. coli clones containing plasmid pDL III-1a which is the pKK223-3plasmid containing a dicistronic loader gene/des-val alpha constructunder the regulation of the P_(tac) promoter would be transformed with aplasmid containing the dicistronic loader gene/des-val beta constructunder control of the same promoter, but with a gene conferringadditional antibiotic resistance to tetracycline. This could beconstructed in the following manner: Plasmid pJR IV-50a contains thedes-val beta globin gene under control of the P_(tac) promoter. Thisplasmid would be cut with PvuII to generate a linear plasmid with bluntends. This would be ligated with a NotI phosphorylated linker (NewEngland BioLabs). The ligation mixture will be used to transform E.coli. Plasmid DNA would be prepared and plasmids containing a NotI siteidentified by digestion with NotI agarose gel electrophoresis. Thisplasmid will contain the P_(tac) :des-val beta globin sequence. The genefor resistance to the antibiotic kanamycin is commercially available(Pharmacia) and contains EcoRI restriction sites on both ends. The endswill be converted to blunt ends by treatment with T4 DNA polymerase bythe method of Maniatis, et al. The resulting fragment will be ligatedwith a 50 fold excess of phosphorylated NotI linker (25° C., 60 min).The ligation reaction would be made 0.01M in EDTA, heated to 70° for 20min and ethanol precipitated. The precipitated DNA will be taken up in100 ul of NotI buffer and treated with 100 units of NotI (37° ) for 2hr. The fragment would be purified by agarose gel electrophoresis. TheNotI adapted kanamycin resistance gene would then be ligated into theNotI linearized pJR IV-50a to yield a plasmid with the gene for des-valbeta globin under control of P_(tac) with kanamycin resistance. E. coliJM-109 clones containing plasmid pDL III-1a, the plasmid containingdes-val alpha globin under control or P_(tac) with resistance toampicillin, will then be transformed with kanamycin resistant plasmidcontaining the gene for des-val beta globin and clones will be selectedfor resistance to both ampicillin and kanamycin. Other antibioticresistance genes could be used as well. Expression of alpha and betaglobin polypeptides under the regulation of separate promoters will thenbe analyzed by IPTG induction, SDS page and western blotting.

Should we encounter problems with plasmid exclusion, we could use thesame strategy with the pIN plasmids that have been used to expresspolypeptides from separate plasmids in E. coli (McNally, et al., PNAS85, 7270, 1988).

One potential problem that we may face with creating plasmids in whichthe alpha and beta globin genes are under separate but identicalpromoters is the possibility of homologous recombination within theidentical sequences on the plasmids, eg. the promoter region. This couldresult in deletion of a segment of important DNA sequence. It istherefore preferable to use different, non-homologous promoter for eachdifferent globin gene, eg. P_(tac) and _(ptrc), or the lambda P_(L)promoter in appropriate host (containing cI857).

EXAMPLE 18 Synthesis and Assembly of the Di-Alpha Beta Globin Constructin aP_(L) Regulated Vector System

In prior examples, the globin genes were under Tac promoter control, andthe alpha (or di-alpha) and beta globin genes were each translationallycoupled to the ribosomal loader cistron taught by Schoner, et al. Inthis example, the lambda P_(L) promoter and a different translationalcoupler (see below) are used.

Translational Coupler

    __________________________________________________________________________      SD2              MET    Sfi                                                 5'                                                                              AAT AAG GAG                                                                           GAA                                                                              TAA                                                                              CAT                                                                              ATG                                                                              CTG                                                                              TCT CGG GCC GAT (Cont'd)                             3'                                                                              TTA TTC CTC                                                                           CTT                                                                              ATT                                                                              GTA                                                                              TAC                                                                              GAC                                                                              AGA GGC CGG CTA (Cont'd)                                                       EAGI                                                AAG GCC CCA AGC TTG GGG3'                                                     TTC CGG GGT TCG AAC CCC5'                                                           Hind III                                                                __________________________________________________________________________

The pL expression system has a different translational coupler ascompared to the pTAC system. Sequences coding an SD and a translationalstop were added downstream from the 5' end of the N protein codingsequences to act as a translational coupler. Subsequent to that theglobin coding sequences are identical to that used in the pTAC system.

Using the pPL-lambda vector available from Pharmacia, a plasmidconstruct was assembled to generate a genetically crossed-linkedtetrameric human hemoglobin (wild type) in E. coli strains N99Ci+ andN4830-1 (cI857). These bacterial strains were obtained from Pharmaciaand are inducable by addition of naladixic acid (40 μg/ml) or mitomycinC (10 μg/ml) in the presence of the wild type Ci+ repressor or heattreatment of the strain containing the cI857 repressor gene (Mott, etal., PNAS 82, 88, 1985 and Gottesmann, M. E., et al., J. Mol. Biol. 140,57, 1980). A diagramatic representation of the cloning strategy isdepicted in FIG. 19.

Removal of the EagI Site from the pPL-Lambda Vector

Removal of the EagI site from pPL-lambda was necessary to enable cloningof the di-alpha gene sequence, because both alpha structural genescontain a EagI site located 6 bp into the coding sequence. ThepPL-lambda vector was digested with EagI, and the ends were filled usingT4 DNA polymerase. The Bam HI linker (5'-CCCGGATCCGGG-3')(Pharmacia),was blunt-end ligated to the EagI digested pPL-lambda plasmid bystandard methods. This eliminated the EagI site in the desiredconstruct. The resulting mixture was digested with EagI to eliminate anyplasmids still containing the EagI site. E. coli N99Ci+ cells weretransformed with resulting plasmid, pPL-lambda-E. Clones containing thedesired plasmid were identified by restriction digest analysis.

Incorporation of the Synthetic Co-Translational Coupler intopPL-Lambda-E

Prior to inserting the globin genes into the vector it was necessary toincorporate the synthetic translational coupler sequence into the HpaIsite of pPL-lambda-E. This was done by digestion of pPL-lambda-E withHpaI followed by blunt-end ligation of the co-translational coupler intothe HpaI site of the vector. Ligation of the coupler to the blunt endresulted in destruction of the HpaI site. The ligation mixture wastreated with HpaI to digest any plasmid remaining containing the HpaIsite. E. coli N99Ci+ cells were transformed with the resulting reactionmixture. Clones were screened with EcoRI and Hind III restrictiondigests to identify clones containing the co-translational coupler inthe proper orientation. DNA fragments of 522 bp and 4762 bp wereobserved for plasmid containing the desired orientation. To confirm theorientation of the coupler, the resulting plasmid was sequenced using aprimer (5'CAATGGAAAGCAGCAAATCC-3') complementary to the sequence 30 basepairs upstream from the translational coupler sequence. The desiredplasmid was denoted as pPL-lambda-E+TC.

Construction of an Expression Plasmid Containing Des-Val Alpha and BetaGenes Under Control of pPL-Lambda

The Des-Val alpha and beta globin genes were obtained from pDL III-14cby digestion with EagI and Hind III, followed by agarose gelpurification of the desired 942 bp segment. The purified alpha and betaglobin gene fragment was cloned into EagI and HindIII digestedpPL-lambda-E+TC. The ligation mixture was used to transform E. coliN99Ci+, and clones were screened for the presence of the desiredplasmid, pPL-alpha/beta. Digestion with EcoRI (4758 and 1468 bp), andwith PstI and HindIII (4005, 1775, 520 bp) confirmed presence of thedesired restriction sites. Further confirmation was obtained throughsequencing with the 20 bp primer, above, to confirm the sequence betweenthe co-translational coupler and the Des-Val alpha globin gene, and asecond primer (5'ACCCGGAAAACTTCCGTC-3') to confirm the sequence betweenDes-Val beta and the pPL vector.

Construction of an Expression Plasmid Containing Di-Alpha and BetaGlobin Genes Under Control of pPL-Lambda

An RGV crosslinker which encodes for the carboxy terminal portion ofalpha globin, linked via a single glycine residue to the native sequenceof the amino portion of a second alpha globin chain, was prepared byseparately phosphorylating the 5' ends of 5'CGAAATAACGTGGTGTTCTGTCTGC-3'and 3'TTTATGGCACCACAAGACAGACGCCGG-5 ' with T4 kinase, followed byannealing. This double stranded oligonucleotide was cloned onto the EagIend of a purified fragment of Des-Val alpha and beta globin preparedfrom pDL III-14c digested with EagI and HindIII, as described above. Thelinear DNA sequence generated from this ligation, now containing stickyends coding for BstBI and HindIII restriction site sequences, waspurified by agarose gel electrophoresis and cloned into BstBI andHindIII digested pPL-alpha/beta. The new plasmid designatedpPL-dialpha/beta contained a sequence with a co-translational couplerupstream to a sequence containing di-alpha globin linked via a glycineresidue, followed by a cotranslational coupler adjacent to a beta globingene sequence, all under control of a single P_(L) promoter. Theseclones were identified through screening minipreps with EagI restrictiondigestions. Clones without the second alpha globin gene merelylinearised upon digestion, whereas clones containing the second genereleased 431 bp and 6222 bp DNA fragments.

Construction of the Expression Plasmid pSGE0.1-L0 Containing aROP-Origin of Replication Mutation

pPL-dialpha/beta was digested with PvuII then treated with T4 DNApolymerase to fill in the sticky ends. The linearized plasmid was thenblunt end ligated witha NotI linker (Promega Corp., Madison, Wis.)(5'TTGCGGCCGCAA-3'). The ligation mixture was then treated with PvuII toremove any remaining plasmid containing the PvuII site. E. coli weretransformed with pSGE0.1-L0 and positive clones were identified by thepresence of the unique NotI restriction site.

Expression of Hemoglobin SGE0.1 in E. coli Under the Control of theLambda P_(L) Promoter

E. coli N99Ci+ and E. coli N4830-1 were transformed with pSGE0.1-L0 andgrown on agar plates containing ampicillin, as described previously.These E. coli strains contain the cI+ repressor gene and the cI857 heatsensitive repressor gene, respectively.

Inocula of N99Ci+ were grown at 37° C. in TB media to an OD₆₀₀ of .sup.˜1.0 and induced with naladixic acid (40 μg/ml). Cultures were incubatedfor 4-6 hrs. at 37° C. before the cells were harvested. Hemoglobinproduction was estimated by SDS-PAGE analysis of total cell protein andby western blot analysis. By these techniques, SGE0.1 was estimated tobe produced at .sup.˜ 0.02% of the total cell protein in this cell line.Hemoglobin (57 μg) was isolated by Mono Q chromatography and shown tohave an optical spectrum representative of that for normal hemoglobin.

Inocula of N4830-1 were incubated at 30° C. in TB media to an OD₆₀₀ of1.0 and induced by addition of sufficient preheated TB media (65° C.) toraise the temperature of the inocula TO 42° C. The culture was thenincubated at 42° for 4-6 hrs. Total cell protein analysis with SDS-PAGErevealed that SGE0.1 was being synthesized at 0.4% of the total cellprotein, corresponding to 0.18 mg protein per gram of wet cell paste.SGE0.1 prepared from a 2 L preparation was purified as describedelsewhere, and resulted in isolation of 7.6 mg of purified material.SGE0.1 (7.6 mg) was isolated and had a P₅₀ of 4.1 (Hemox Analyzer, pH7.4, 0.1M NaCl, 37° C.), and an optical spectrum representative ofnative hemoglobin.

EXAMPLE 19 Production of Hemoglobin in Yeast

All restriction enzymes and DNA-modifying enzymes were purchased fromBRL, New England Biolabs, IBI, Pharmacia or Boerhringer-Mannheim. Theconcentrations of enzymes used were those suggested by the supplier toproduce a complete reaction in 30 minutes. The buffers and conditionsfor the use of these enzymes were those provided with the enzymes,unless otherwise stated. Plasmid DNA was purified from E. coli DH5α asdescribed by Birnboim and Doly (Nucleic Acids Research 1979,7:1513-1520). Electrophoretic analysis of DNA was carried out in agarosegels using tris-acetate electrophoresis buffer (Maniatis et al.Molecular Cloning, Cold Spring Harbor, N.Y., 1982). DNA was visualizedby staining the gels with 0.5 μg/ml ethidium bromide and exposing thegel to ultraviolet light. DNA fragments were purified from agarose gelsusing a kit purchased from BIO-101. DNA fragments were purified fromacrylamide gels by crushing the excised gel fragment, containing the DNAof interest, in 3.25M ammonium acetate and incubating overnight at 37°C. Gel fragments are removed by centrifugation (12,000×g, 15 min) andthe DNA precipitated with 2 volumes of 95% ethanol, 5% isopropanol. Theprecipitate is dried in vacuo and dissolved in 0.1×TE (1×TE is 10 mMTris. HCl pH 7.8, 1 mM Na₃ EDTA). Acrylamide gel electrophoresis of DNAwas done as described by Maniatis, et al. (Molecular Cloning, ColdSpring Harbor, N.Y., 1982.) in tris-acetate electrophoresis buffer.Bacteriological growth media and DNA transformation methods aredescribed by R. W. Davis et al. (Advanced Bacterial Genetics, ColdSpring Harbor Laboratory, New York, 1980, p140-141). Transformation ofS. cerevisiae with linear or circular DNA was carried out as describedby H. Ito et al. (J. Bacteriology 153:163-168 (1983)). Transformantswere selected on SD medium lacking uracil or tryptophane (SD-ura,SD-trp) depending on the selectable marker on the plasmid (F. Sherman etal., Methods in Yeast Genetics: A Laboratory Manual, Cold Spring HarborLaboratory, 1979). All other yeast media used are described by Shermanet al. (ibid.)

SYNTHESIS AND ASSEMBLY OF A GALACTOSE REGULATED PROMOTER

This synthetic promoter consists of two functional parts, a regulatorysequence and sequence that allows efficient initiation of mRNAsynthesis. One of the regulatory regions we chose includes thenucleotide sequence that confers positive regulation of transcription inthe presence of galactose (M. Johnston and R. Davis, 1984. Molecular andCellular Biology 4:1440-1448; L. Guarente et al., 1982, Proc Nat AcadSci (U.S.A.) 79:7410-7414.). The transcriptional initiation site isderived from the consensus sequence for the S. cerevisiaeglyceraldehyde-3-phosphate dehydrogenase gene (GAP491) (L. McAlister andM. J. Holland, J. Biol Chem 260:15019-15027, 1983; J. P. Holland et al.,J. Biol Chem 258:5291-5299, 1983).

The synthetic oligonucleotides shown in FIG. 1 were synthesized on aBiosearch 8600 DNA synthesizer. Each oligonucleotide was cleaved fromits support column with 28% NH₄ OH. The blocking groups were removed byincubating the cleaved oligonucleotide in 28% NH₄ OH at 65° C. for ≧16hr. All oligonucleotides were purified by preparative polyacrylamide gelelectrophoresis in slabs of 10% acrylamide (19:1acrylamide:bis-acrylamide) containing 7M urea. oligonucleotides wereeluted from acrylamide slices by incubation (16 hr) in 50 mM ammoniumacetate (pH 7.4) 2.5 mM magnesium acetate, 0.25 mM EDTA and 0.25% SDS at37° C. Acrylamide fragments were removed by centrifugation (14,000×g, 10min) and the oligonucleotide precipitated from the aqueous phase by theaddition of NaCl to 0.25M and 3 volumes of 100% ethanol. Theprecipitated oligonucleotides were collected by centrifugation (14,000×gfor 30 min), washed twice with 80% ethanol, once with 100% ethanol andthe pellets dried. Pellets were dissolved in 0.1×TE. 2×10⁻¹⁰ moles(each) of oligonucleotides 1-5 (Table 5) were phosphorylated in 0.02 mlof 0.066M Tris HCl (pH 7.6), 0.01M MgCl₂, 0.002M dithiothreitol (DTT),0.001M spermidine and 10 units of T4 polynucleotide kinase.Phosphorylation reactions were carried out at 37° C. for 30 min andterminated by heating to 96° C. for 5 min. 2×10⁻¹⁰ moles ofoligonucleotides 1 and 6 (Table 5) were added to the phosphorylatedoligonucleotides in a final volume of 0.0.4 ml or 0.07M Tris HCl (pH7.6) 0.01M MgCl₂. The mixture of oligonucleotides 1-6 (Table 5) washeated to 96° C. for 5 min, 2×10⁻¹⁰, 75° C. for 20 min, 55° C. for 30min, 37° C. for 60 min and 25° C. for 15 min. T4 DNA ligase (10 units),DTT (0.002M final concentration), and ATP (0.01M) were added and themixture incubated at 4° C. for 16 hr. The resulting 210 bpoligonucleotide contains a 5' end compatible with a SalI restrictionendonuclease site and a 3' end compatible with an XbaI site. Because theoligonucleotides comprising the two ends of the intact oligonucleotidewere not phosphorylated, they cannot ligate to each other. Thisoligonucleotide was cloned into the vector pSK+ (Stratagene, Inc.) (FIG.21(a)) that had been digested with XbaI and SalI. The ligation mixturecontained 50 ng of XbaI, SalI digested pSK(+), and 5 pMoles of theligated oligonucleotide in a volume of 0.01 ml. E. coli DH5α wastransformed with a portion of the ligation reaction and clones thatcontain inserts were identified by screening for white colonies onLB-ampicillin (0.15 mg/ml) agar plates supplemented with XGAL (4 μg/ml).Positive identification was made by preparing plasmid DNA from theseisolates and digesting with XbaI and SalI. The restriction digests wereanalyzed by agarose gel electrophoresis and three clones containing afragment of the expected size (.sup.˜ 250 bp) were identified. The DNAsequence of all three clones was determined and one was chosen forfurther use and designated pGS2488 (FIG. 21(a)).

ASSEMBLY OF THE SYNTHETIC GALACTOSE UPSTREAM ACTIVATOR (GAL_(UAS))SEQUENCE

The oligonucleotides shown in Table 6 were synthesized and purified asdescribed above. Oligonucleotides 2-5 were phosphorylated, annealed witholigonucleotides 1 and 6 and ligated as described for the assembly ofGAP. The full length oligonucleotide generated by this protocol hasnon-phosphorylated ends compatible with the restriction endonucleasesites generated by SphI and SalI. However, when the oligonucleotide isligated to a SalI site the resulting junction formed between the twofragments will no longer contain a cleavable SalI site.

The GAL_(UAS) is contained on an SPHI-SalI fragment. To clone thisfragment into pGS2488 required that we change the KpnI site of thisplasmid to an SphI site. The plasmid pGS2488 was modified by cleavingwith KpnI. The KpnI digested plasmid was incubated with 2 units of T4polymerase in 0.05 ml buffer containing 50 μM of eachdeoxyribonucleotide triphosphate (A,G,C,T), 0.033M Tris-acetate (pH7.9), 0.066M potassium acetate, 0.01M magnesium acetate, 0.5 mM DTT and100 μg/ml bovine serum albumin (BSA). Na₃ EDTA was added to 0.015M andthe mixture extracted 1× with phenol-chloroform. DNA was precipitatedwith ethanol. The dry pellet was dissolved in 0.008 ml of T4 DNA ligasebuffer, 50 ng phosphorylated SphI linkers (New England Biolabs) and 10units of T4 DNA ligase. The mixture was incubated for 1 hour at 25° C.and used to transform E. coli DH5α. Plasmid DNA was prepared from 12transformants and tested by restriction enzyme digestion and agarose gelelectrophoresis, for the presence of an SphI site and the absence of aKpnI site. A clone containing a plasmid with these characteristics wasidentified and the plasmid was designated pGS2888 (FIG. 21(b)).

The next step in the assembly of this hybrid promoter was to clone theSphI-SalI fragment containing the GAL_(UAS) into pGS2888. pGS2888 wasdigested with SphI and SalI, phenol-chloroform extracted and ethanolprecipitated. Fifty nanograms of SphI, SalI digested pGS2888 wasincubated with 25ng of the annealed, ligated GAL_(UAS) mixture in 0.005ml 1× ligase buffer containing 10 units of T4 DNA ligase. The ligationmixture was incubated overnight at 4° C. and a portion used to transformE. coli DH5α. Ampicillin resistant clones were isolated and plasmid DNAprepared. The plasmid DNA (digested with XbaI and SpI) was analyzed byagarose gel electrophoresis. A plasmid containing a fragment of theexpected size (.sup.˜ 500 bp) was identified. The sequence of theputative GAL_(UAS) portion of this plasmid was determined and theplasmid was designated pGS4788 (FIG. 21(b)). The complete sequence ofthe synthetic GALGAP promotor (pGGAP) is shown in FIG. 20.

CONSTRUCTION OF A pGGAP-β-GLOBIN EXPRESSION CASSETTE

The plasmid pLCIIFX-β-globin (K. Nagai, M. Perutz and C. Payart, ProcNat Acad Sci (U.S.A.) 82:7252-7255) was used as the source of humanβ-globin cDNA. The coding region of the β-globin cDNA can be excised asan ApaL1 to Hind3 fragment that is missing only the first fournucleotides of the β-globin coding (translated) sequence. pLcIIFXβ-globin (5 μg) was digested with ApaL1 and Hind3 and a 550 bp fragmentcontaining the cDNA was purified by acrylamide gel electrophoresis. TheApaL1-Hind3 fragment containing the β-globin cDNA was cloned into Xba1,Hind3 digested pUC19 using the following adaptor (synthesized andpurified as described above):

XbaI NcoI ApaL1 YH1a/b5'-CTAGAACCATGG TTGGTACCACGT-5'

The two oligonucleotides were mixed (12.6 μg of each), NaCl was added to0.25M and three volumes of ethanol (anhydrous) was added. Theprecipitated oligonucleotides were collected by centrifugation(14,000×g, 15 min) and the pellet washed twice with 80% ethanol, oncewith anhydrous ethanol and dried in vacuo. The adaptors were dissolvedin 1 ml of 0.1×TE. Ligation rections were conducted using fiftynanograms of Xba1-Hind3 fragments containing the β-globin cDNA and 12.5ng of the ethanol precipitated (non-phosphorylated)YH1a.holigonucleotide in a total of 10 μl of T4 DNA ligase buffer containing10 units of T4 DNA ligase. The ligation mixture was incubated at 4° C.for 16 hr. and a portion used to transform E. coli DH5α. Transformantswere selected on LB-ampicillin plates containing 4 μg/ml XGAL. PlasmidDNA was prepared from 12 white colonies. The DNA was digested with Nco1or ApaL1 and analyzed by agarose gel electrophoresis. Four of thesecolonies contained plasmids with the expected restriction fragments andone was designated pUC19β-globin (FIG. 21(a)). The plasmid pSUC2-6Σ (G.Stetler et al. Biotechnology 7:55-60 (1989)) (FIG. 21(a)) was digestedwith Hind3 and Xba1 and the large fragment was purified by agarose gelelectrophoresis. The Xba1 to Hind3 fragment containing β-globin cDNA wasalso purified (agarose gel electrophoresis) from pUC19β-globin. Tennanograms of gel-purified Xba1 , Hinds digested pSUC2-εΣ was mixed with16 nanograms of the Xba1, Hind3 fragment from pUC19β-globin in 0.01 mlof ligase buffer containing 10 units of T4 DNA ligase. The ligationmixture was incubated at 25° C. for 1 hr. and a portion used totransform E. coli DH5α. Transformants were selected on LB-ampicillinmedium and three were used to prepare plasmid DNA. These were analyzedby digestion with EcoR1 and analyzed by agarose gel electrophoresis. Twoof these contained plasmids with the expected restriction fragments andone was designated pGS1188 (FIG. 21(b)). This plasmid contains β-globinunder the transcriptional control of the sigma promoter and contains the3' transcriptional termination signals and polyadenylation signals ofthe MFα1 gene.

REPLACEMENT OF THE SIGMA PROMOTER WITH pGGAP

The plasmid pGS1188 was digested with SphI and NcoI and the vector plusβ-globin cDNA was separated from the sigma promoter by agarose gelelectrophoresis and purified as described previously. Plasmid pGS4788(10 μg) was also digested with SphI and NcoI and the .sup.˜ 500 bpfragment (containing pGGAP) produced by this digest was purified byagarose gel electrophoresis. Fifty nanograms of the gel-purifiedβ-globin containing vector was incubated with 50 ng of NcoI-SphIfragment containing the pGGAP promotor in 0.01 ml of 1× ligase bufferwith 10 units of T4 DNA ligase. The mixture was incubated for 1 hour at25° C. and a portion of the ligation mixture used to transform E. coliDH5α. Ampicillin resistant clones were selected. Plasmid DNA isolatedfrom 12 of these clones was analyzed by digestion with SphI and NcoI toidentify plasmids containing the .sup.˜ 500 bp GGAP promoter. Thepresence of the β-globin cDNA was confirmed by digestion with XbaI andSalI, followed by agarose gel electrophoresis analysis. A plasmidcontaining the expected fragments was identified and designated pGS3588(FIG. 21(b)). To aid the subcloning of this fragment into a yeastvector, the Smal site of pGS3588 was converted to a XhoI site asfollows: 1 μg of pGS3588 was digested with SmaI, the digest wasextracted once with phenol-chloroform and ethanol precipitated. Theprecipitated DNA was dissolved in T4 DNA ligase buffer, with 100 ng ofphosphorylated XhoI linker and 10 units of T4 DNA ligase (final volumeof 0.01 ml). The ligation was incubated at room temperature for 2 hr anda portion of the ligation mixture was used to transform E. coli DH5α(excess linkers were not removed prior to transformation). Ampicillinresistant clones were isolated and plasmid DNA prepared. Plasmidscontaining the additional XhoI site were identified by digestion withXhoI and agarose gel electrophoresis analysis. A plasmid containing thepGGAP-β-globin expression cassette was identified and has beendesignated pGS3888 (FIG. 21(b)).

CONSTRUCTION OF AN α-GLOBIN EXPRESSION CASSETTE

We obtained a partial length cDNA (pα-MRC) clone from K. Nagai (MRC,Cambridge). To adapt the cDNA encoding α-globin for expression from thepGGAP promotor, two oligonucleotide primers were synthesized (synthesisand purification of oligonucleotides was as described above).

α-1: 5'-GAATTCCATGGTGCTGTCTCCTGCCGACAAGACC-3'.

α-2: 5'-CTGCAGTCGACTTAACGGTATTTGGAGGTCAGCACGGTGCT-3'.

These two oligonucleotides were used as primers for a polymerase chainreaction (PCR) (R. K. Sakai et al., 1985. Science 230:1350-1354) using aPerkin Elmer-Cetus PCR kit and pα-MRC as template. α-globin cDNA (8.3ug/ml) in 0.018 ml H₂ O was denatured by the addition of 0.005 ml 10MNaOH. The mixture was incubated at 25° C. for 5 min. Denatured DNA wasprecipitated by the addition of 0.003 ml 3M sodium acetate (pH 5.2) and0.075 ml anhydrous ethanol. The precipitated DNA was washed twice with80% ethanol, once with 100% ethanol and dried. The dried pellet wasdissolved in: 0.005 ml 20 μM α-1, 0.005 ml 20 μM α-2, 0.010 ml 10× Taqpolymerase buffer (Perkin Elmer-Cetus), 0.0005 ml TaqI polymerase(Perkin Elmer-Cetus), 0.663 ml H₂ O, 0.016 ml of all fourdeoxyribonucleotide-triphosphates (1.25 mM each). TaqI polymerase wasadded after heating the solution to 94° C. for 1 min. After addition ofthe enzyme the aqueous solution was overlayed with 0.100 ml paraffinoil. The reaction-mixture was cycled, by hand, 25 times at the followingtemperatures: 37° C. for 2 min, 68.5° C. for 3 min and 94° C. for 1 min.After the twenty-fifth cycle, the reaction mix was incubated at 37° C.for 2 min and 68.5° C. for 10 min. A portion (0.005 ml) of the reactionmixture was analyzed by agarose gel electrophoresis and revealed a bandof the expected size (.sup.˜ 430 bp). The PCR-amplified DNA fragmentshould contain a 5' extension, that includes an ATG codon embedded in anoptimal sequence context for initiation of translation (M. Kozack 1986,Cell 44:283-292). The 3' end should contain an extension that includes atranslational terminator and a SalI restriction endonuclease site. ThePCR-amplification reaction was phenol-chloroform extracted and ethanolprecipitated. The dry pellet was dissolved in 0.05 ml 1 mM Tris-HCl (pH7.8). A portion of this material (0.025 ml, 1.25 ug) wad digested withNcoI and SalI and purified by acrylamide gel (5%) electrophoresis. A gelslice containing the fragment was eluted by crushing the gel slice in0.3 ml 2.5 ammonium acetate (pH 7.4) and incubating at 37° C. for 16 hr.Acrylamide fragments were removed by centrifugation and the DNAprecipitated by the addition of 0.75 ml of ethanol. The pellet wascollected and dissolved in 0.020 ml 1mM Tris HCl (pH 7.8) 0.1 mM EDTA.This fragment was cloned into NcoI, SI digested and agarose gel purifiedpGS3888. Fifty nanograms of NcoI, SalI digested pGS3888 was incubated (2hr, 25° C.) with 50 ng of gel purified, PCR-amplified NcoI-SalI fragmentcontaining the α-globin cDNA in 0.01 ml ligase buffer with 10 units ofT4 DNA ligase. A portion of this reaction mixture was used to transformE. coli DH5α- and ampicillin resistant clones were selected onLB-ampicillin medium. Plasmid DNA was prepared from 12 independentisolates and digested with Nco1 and SalI. The restriction digests wereanalyzed by acrylamide gel electrophoresis (5%), all twelve contained afragment of the expected size. One of these was designated pGS4088 (FIG.22). The α-globin insert in pGS4088 was completely sequenced to assurethat no mutations had been introduced by PCR-amplification.

CONSTRUCTION OF A YEAST EXPRESSION PLASMID THAT CO-EXPRESSES α ANDβ-GLOBIN GENES FROM A SINGLE PLASMID

The α-globin and β-globin expression cassettes from the plasmids pGS3888and pGS4088 were cloned into a single plasmid in a way that allows themto be excised on a single NotI fragment. This NotI fragment was thencloned into the high copy yeast plasmid pC1N to generate a plasmidcarrying and expressing both α- and β-globin chains under the control ofseparate (though identical) promoters. The details are presented below.

INTRODUCTION OF NOTI SITE INTO pSK(+)

The plasmid pSK(+) (Stratagene, Inc.) (FIG. 23(a)) was modified bydigesting 100 ng of purified plasmid DNA with KpnI. After digestion wascomplete, the DNA was ethanol precipitated and the dry pellet dissolvedin 0.05 ml T4 DNA polymerase buffer containing 10 units of T4 DNApolymerase. The reaction mixture was incubated at 37° C. for 20 min, Na₃EDTA (10 mM) was added and the sample heated to 70° C. for 10 min. Thedigested DNA was precipitated with ethanol and the dry pellet dissolvedin 0.01 ml of 10 mM Tris-HCl (pH 7.8) 1 mM EDTA. A portion of thismaterial (20 ug) was dissolved in 0.005 ml of ligase buffer containing10 units of T4 DNA ligase and 50 ng of phosphorylated NotI linkers. Theligation mixture was incubated at 25° C. for 2 hr and a portion used totransform E. coli DH5α. Ampicillin resistant colonies were selected onLB-ampicillin medium. Plasmid DNA was isolated, digested with Not1 andanalyzed by acrylamide gel (5%) electrophoresis. A plasmid containingthe additional NotI site is expected to generate a new, .sup.˜ 90 bpfragment. Such a plasmid was identified and designated pSN(+) (FIG.23(a)).

CLONING THE pGGAP-α-GLOBIN EXPRESSION CASSETTE INTO pSN(+)

pSN(+) was digested with SalI, phenol-chloroform extracted and ethanolprecipitated. The precipitated DNA was dissolved (50 ug/ml) in 1 mMTris-HCl (pH 7.8), 0.1 mM EDTA. pGS4088 was digested with XhoI and the.sup.˜ 1100 bp fragment containing the α-globin expression cassette wasisolated from an agarose gel. The purified fragment was dissolved (20ug/ml) in 0.1 L×TE. Twenty-five nanograms of SalI digested pSN(+) wasmixed with 50 ng of the gel-purified α-globin fragment in 0.01 ml ligasebuffer containing 10 units of T4 DNA ligase. The ligation mixture wasincubated for 1.5 hr at 25° C. and a portion used to transform E. coliDH5α. Ampicillin resistant clones were isolated. One hundredtransformants were transferred, in a grid pattern, to fresh plates.Replicas of the grid were made on a nitrocellulose filter and preparedfor colony hybridization (R. W. Davis et al. Adv Bacterial Genetics,Cold Springs Harbor, N.Y., 1980.). The hybridization probe wasoligonucleotide 4 (Table 6). This oligonucleotide (20 pM) was labelledwith ³² P-ATP in a 0.02 ml reaction mixture containing 0.050M Tris-HCl(pH 7.6), 0.01M MgCl₂, 0.005M DTT, 0.0001M spermidine, 0.0001M EDTA, 50pM of ³² P-ATP and 10 units of T4 polynucleotide kinase. The reactionmixture was incubated for 2 hr at 37° C., unincorporated ATP was removedby spin-column chromatography (BIORAD) used according to themanufactures instructions. The filters were incubated (37° C.) inhybridization solution (6×SSC, 50% formamide, 2% SDS, 20 pMoles of ³² Plabelled probe) for 16 hr. The filters were washed 4 times (15 min each)with 1×SSC, 0.1% SDS at 55° C.; twice (5 min each) with 0.2×SSC, 0.1%SDS at 50° C. and twice (5 min each) with. 0.2×SSC. Autoradiographs weremade of the filters. Colonies that produced a hybridization signal wereused to prepare plasmid DNA. This DNA was digested with EcoR1 and thefragments produced by this digest were analyzed by agarose gelelectrophoresis. A plasmid containing a fragment of the correct size wasidentified and designated pGS4888 (FIG. 23(a)).

CLONING THE pGGAP-β-GLOBIN EXPRESSION CASSETTE INTO pGS4888

The construction of pGS4888 required cloning an XhoI fragment(pGGAP-α-globin) into a SalI site. The combination of a XhoI site with aSalI site destroys the recognition sites for both XhoI and SalIrestriction endonucleases, leaving pGS4888 with a single unique XhoIsite. An XhoI fragment from pGS3888 was purified by agarose gelelectrophoresis and cloned into XhoI digested pGS4888 essentially asdescribed for the construction of pGS4888. Colony filter hybridizationswere performed using the NcoI-SalI fragment (containing β-globin cDNA)from pGS3888 as a hybridization probe. This fragment was purified byagarose gel electrophoresis and radioactively labelled with α³² P-dCTPusing an Amersham random oligonucleotide labelling kit. Filters werehybridized, washed and autoradiographs made as described for theconstruction of pGS4888. Plasmid DNA was prepared from coloniesproducing a hybridization signal and digested with either NcoI or NcoIand SphI. These digests allow identification of plasmids that containboth the α- and β-globin expression cassettes and reveals the relativeorientation of the α- and β-globin transcriptional units. A plasmidcontaining both transcriptional units was identified and given thedesignation pGS189 (FIG. 23(b)).

CONSTRUCTION OF PLASMID pC1N

The plasmid pC1U was modified to introduce a NotI site. pC1U DNA (10 ug)was digested with SalI, phenol-chloroform extracted and ethanolprecipitated. The SalI ends were modified with T4 DNA polymerase toproduce "blunt" ends and phosphorylated NotI linkers were added. Theprocedures used for these modifications are essentially the same asthose used to produce pSN(+) (described above). The plasmid resultingfrom these manipulations is called pCIN (FIG. 23(b)). This pC1N wasdigested with Notl, phenol-chloroform extracted and ethanolprecipitated. A .sup.˜ 2.4 kb NotI fragment (carrying α- and β-globinexpression cassettes) was purified from NotI digested pGS189 DNA byagarose gel electrophoresis. Fifty nanograms of NotI digested pC1N wasmixed with 50 ng of the gel purified, 2.4 kb fragment isolated frompGS189 in 0.01 ml of ligase buffer containing 10 units of T4 DNA ligase.The reaction mixture was incubated at 25° C. for 2 hr and a portion usedto transform E. coli DH5α. Ampicillin resistant clones were selected onLB-ampicillin plates and plasmid DNA prepared. The purified DNA wasdigested with EcoR1 and analyzed by agarose gel electrophoresis toidentify plasmids carrying the α- and β-globin genes and to determinethe orientation of the insert with respect to the vector. Two plasmidswere identified, representing the two possible orientations and havebeen designated pGS289 (FIG. 23(b)) and pGS389 (FIG. 23(b)).

EXPRESSION OF RECOMBINANT HUMAN HEMOGLOBIN IN SACCHAROMYCES CEREVISIAE

S. cerevisiae strains GSY112 (MATαpep4::H1S3prb1 1.6R his3 200 ura3-52leu2::hisG can1 cir°) and RSY334 (MATα- reg1-501 pep4-3 prb1-1122ura3-52 leu2-3, leu2-112) were transformed with plasmids pGS289 orpGS389 by the method of Ito et al. (J. Bacteriology 153:163-168 (1983)).Transformants were selected on yeast SD-ura. Single colony isolates werepicked and streaked to SD medium lacking uracil and leucine. Coloniesfrom this selective medium (SD-ura,-leu) were used to inoculate 2 ml ofSD-ura,-leu. The cultures were incubated at 30° C. for 24 hr and used toinoculate 20 ml cultures of YP+3% ethanol. These were incubated for anadditional 24 hr and galactose was added to a concentration of 2%.Samples were removed at 4, 8, 24, 28, 32 and 48 hours post induction.Cells were collected by centrifugation and washed with 1 ml 10 mM TrisHCl (pH 7.8), 1 mM EDTA and resuspended in SDS-PAGE sample buffer (2×10⁸cells/ml). Samples were boiled for 10 min and the insoluble materialremoved by centrifugation. Samples 0.005 ml were analyzed by SDS-PAGE(12.5% gel) followed by transfer to nitrocellulose. α- and β-globinchains were stained using commercially available rabbit anti-humanhemoglobin and an immunoblotting kit purchased from Promega. Theprotocols used were supplied by Promega. The immunoblot indicated thatboth chains are synthesized and that pGS289 produces slightly lessmaterial than pGS389. An apparent lack of stoichiometry between theα-chain (lower band) and β-chain is due to a difference inimmunoreactivity of the antibody to the two chains. This wasdemonstrated by comparing Commassie brilliant blue stained gels ofpurified human and recombinant hemoglobin with immunoblotted samples.

EXAMPLE 20 Characterization Of Human Recombinant Hemoglobin Synthesizedin Yeast

RSY334 pGS389! was grown in 100 ml SD-leucine to an OD₆₀₀ of 2.4. Thiswas used to inoculate 1 L of YPE. This culture was shaken at 30° C., for24 hr at which time 50 ml of 40% galactose was added. The incubation wascontinued and the cells harvested 24 hr later (OD₆₀₀ of 9). The cellpellet was resuspended in 100 ml of 0.01M Tris-HCl (pH 7.18), 0.001MEDTA and carbon monoxide bubbled through the cell suspension for 3 min.The cells were collected by centrifugation and after the CO treatmentwere distinctly red. The cell pellet (19 gm) was resuspended in 19 ml oflysis buffer (0.01M NaPO₄ pH 6.0, 0.020M DTT, 1% Triton X 100, 0.001MPMSF, 0.005M benzamidine and 0.06 mM leupeptin) and bubbled with carbonmonoxide for 2 min. The cell suspension was sonicated with Branson 250sonicator equipped with a 0.5 inch disrupter horn. The sonication timewas 2 min (0.5 sec pulses) at full power. This was repeated 4 times with2 min cool-down periods between sonication intervals. The cell debriswas removed by centrifugation (27,000×g for 15 min) and the supernatantsaved (0° C.). The pellet was resuspended in 5 ml of lysis buffer. Theresuspended pellet was sonicated with the microtip as described above(70% of maximum output). The cell debris was removed by centrifugationas described above and this supernatant combined with the first. Thecombined solutions' were clarified by centrifugation (38,000×g, 20 min),producing a clear, red solution. This was loaded (after adjusting the pHto 6.0, with 10 mM phosphoric acid) onto a 5 ml S-Sepharose fast flowcolumn equilibrated with 0.01M sodium phosphate (pH 6.0). All of the redmaterial bound to the column and the column was washed with 20 ml of0.01M sodium phosphate (pH 6.0). The column was eluted with 0.05M sodiumphosphate (pH 7.5), 0.1M NaCl and 1 ml fractions were collected. The redcolor eluted in two fractions. The purity of this material was analyzedby SDS-PAGE and appears to be ≧50% pure after the first chromatographystep. This material was dialyzed against 10 mM sodium phosphate (pH6.0), 0.001M EDTA for 16 hr at 0° C. and rechromatographed on a mono-SFPLC column (ph 6.8-pH 9.0, 0.02M sodium phosphate gradient). The peakfraction from this is ≧85% pure. An absorption spectrum was obtained byscanning the mono-S purified material from 400 mM to 650 mM with aShimadzu spectrophotometer (FIG. 24, top). The spectrum obtained wasidentical to that of human hemoglobin (FIG. 24, bottom), indicating thatthe protein had folded and incorporated heme. The amount of hemoglobinrecovered in the two peak fractions was determined from the extinctioncoefficient (1.23×10⁴ A450/M) to be .sup.˜ 20 mg.

EXAMPLE 21 Construction of Vectors for the Expression of α- and β-Globinfrom Separate Yeast Plasmids

In addition to the development of a single yeast vector that carriesboth α- and β-globin expression cassettes we also developed a systemthat uses separate plasmids for each of the two globin cDNA's. The twoplasmids each carry two yeast genes that are used to maintain theplasmid in yeast. Both have the LEU2 gene in common and one (pGS4688)has the URA3 gene, the other (pGS4988) has the TRP1 gene. By using ahost that carries mutations in URA3, LEU2 and TRP1 both plasmids (onewith α-globin and the other with the β-globin expression cassette) canbe maintained. The constructions of these vectors is described below.

CONSTRUCTION OF pC1T

The plasmid pC1U (5 ug) was digested with BamHI and SalI and the largestfragment was purified by agarose gel electrophoresis. The plasmid YRp7(10 ug) (J. Strathern, E. Jones, and J. Broach, The Molecular Biology ofthe Yeast Saccharomyces, Cold Spring Harbor, N.Y., 1981) was digestedwith BglII and SalI and the fragment containing the TRPI gene waspurified by agarose gel electrophoresis. Twenty-five ng of gel-purified,BamHI and SalI digested pC1U was mixed with 50 ng of the BglII-SalIfragment in ligase buffer containing 10 units of T4 DNA ligase. Thereaction mixture was incubated at 25° C. for 1.5 hr and a portion of theligation reaction mixture used to transform E. coli DH5α. Tetracyclineresistant colonies were selected on LB-tetracycline medium. Plasmid DNAwas prepared from 15 transformants, digested with EcoR1 and analyzed byagarose gel electrophoresis. One isolate with the expected EcoRIrestriction fragments was chosen and was designated pC1T (FIG. 22(a)).

CLONING THE α-GLOBIN EXPRESSION CASSETTE IN pC1T

One hundred nanograms of pC1T was digested with SalI, phenol extracted,ethanol precipitated and dissolved in 0.01 ml of 10 mM Tris-HCl (pH7.8), 1 mM EDTA. The plasmid pGS3888 was digested with XhoI and the.sup.˜ 1.2 kb XhoI fragment containing the β-globin expression cassettewas purified by agarose gel electrophoresis. Ten nanograms of SalIdigested pC1T was mixed with 60 ng of the XhoI fragment containing theβ-globin expression cassette in 0.01 ml of ligase buffer containing 10units of T4 DNA ligase. The reaction mixture was incubated at 25° C. for30 min. A portion of this material was used to transform E. coli DH5α.One hundred ampicillin resistant transformants were picked and patchedto LB-ampicillin agar. They were incubated for 5 hr at 37° C. andoverlayed with a nitrocellulose filter. Plasmids containing the XhoIfragment were identified by colony hybridization, using the XhoIfragment as a hybridization probe, as described above. Coloniesproducing an autoradiographic signal were used to prepare plasmid DNA.The purified DNA was digested with EcoR1 and analyzed by agarose gelelectrophoresis for the presence of the expected restriction fragments.Two clones containing the desired XhoI insert were identified and theorientation of the insert was determined by agarose gel analysis ofEcoR1 digests or SphI digest of the plasmid DNA. Both isolates containedthe desired insert in the same orientation and one was designatedpGS4988 (FIG. 22(a)).

CLONING α-GLOBIN EXPRESSION CASSETTE IN pC1U

The Xhol fragment from pGS4088 containing the α-globin expressioncassette was purified by agarose gel electrophoresis and the recoveredfragment dissolved (100 ng/0.01 ml) in 1 mM Tris-HCl pH 7.8, 0.1 mMEDTA. Twenty-five nanograms of the gel purified fragment was mixed with10 ng of SalI digested (phenol-chloroform extracted, ethanolprecipitated) pC1U in 0.01 ml ligase buffer containing 10 units of T4DNA ligase. The reaction mixture was incubated at 25° C. for 1.5 hr anda portion used to transform E. coli DH5α. Ampicillin resistant colonieswere selected on LB-ampicillin plates. Plasmid DNA was prepared from 12transformants, digested with HindIII and analyzed by agarose gelelectrophoresis. The two possible orientations were isolated and giventhe designation pGS4488 (FIG. 22(b)) and pGS4688 (FIG. 22(b)).

CONSTRUCTION OF DIPLOID STRAINS EXPRESSING α- AND β-GLOBIN CHAINS FROMSEPARATE PLASMIDS

S. cerevisiae RSY330 (MATα pep4-3 prb1-112 hist7 ura3-52 trp1-289 can1gal1) was transformed (Ito et al.) with pGS4488 or pGS4688. Trp⁺transformants were selected on SD-trp medium and streaked for singlecolonies. S. cerevisiae BJY1991 (MATα prb1-112 pep4-3 leu2-3,112trp1-101 ura3-52 gal2 can1) was transformed with pGS4988. URA⁺transformants were selected on SD-ura medium and streaked for singlecolonies. These strains were each tested for the production of α- andβ-globin as follows: (1) Single colonies were picked from SD-selectivemedium and used to inoculate 2 ml of SD-selective (liquid) medium. Thecultures were incubated for 24 hr at 30° C. and diluted into 15 ml odfresh SD-selective medium and incubated for an additional 24 hr. Thecells were collected by centrifugation and resuspended in 25 ml ofYP-galactose (2%) and the incubation continued for an additional 24 hr.The cells were harvested (8,000×g, 10 min) and the pellet washed with 50ml 0.010M TrisHCl(pH 7.8), 0.001M EDTA. The pellets were dissolved byheating to 96° C. for 10 min in SDS-PAGE sample buffer and the debrisremoved by centrifugation (15,000×g, 10 min). The cleared supernatantsfrom 1×10⁶ cells each were analyzed by SDS-polyacrylamide gelelectrophoresis and Western immunoblotting as described above. β-globinwas readily detectable in extracts from BJY3505 pGS4988!. We were alsoable to detect α-globin cross-reacting material, although the signalstrength was considerably weaker. The production of tetramerichemoglobin requires the presence of both α and β chains, ideally thesewould be expressed in the same cell. Because strains BJY3505, BJY1991and RSY330 are haploids they each can be mated with a yeast strain ofthe opposite mating type. Strains RSY330 and BJY1991 are both matingtype α, whereas BJY3505 is mating type a. BJY3505 pGS4988!, RSY330 4688!or BJY1991 4688! or RSY330 pGS4988! matings were done and diploidsselected by streaking onto SD minimal medium with no additional aminoacids or other nutrients. Neither of the plasmid-bearing parentalstrains are capable of growth on this medium, diploids, however, cangrow. Because the diploids are homozygous for the mutations in TRP1 andURA3 both plasmids must be present for the cells to grow in the absenceof these nutrients. These diploid strains were analyzed for thesynthesis of α and β-globin as described above. A most surprising resultwas obtained. Although α-globin and β-globin are synthesized at lowlevels in the haploid strains, co-expression in a diploid strain resultsin a substantial increase in the levels of both chains. Furthermore,after induction (24 hr) with galactose the cell pellets develop adistinct, pink-red color. These results suggest that: (a) co-expressionof α- and β-globin stabilizes the two proteins, perhaps as a consequenceof their interaction and (b) the protein is apparently folding andincorporating heme.

EXAMPLE 22 Oxygen Binding Properties of Yeast-Derived Hemoglobin

The P₅₀ of the yeast derived hemoglobin is nearly identical to that ofHgb Ao. Depending on how we measure it, the P₅₀ ranges. from about 5 to10 torr in a phosphate free solution. At 25° C. it is about 4-6 torr in50 mM BisTris, pH 7.4, NaCl 0.1M. In the same solution, at 37° C., theP₅₀ is from 8.5 to 11.

EXAMPLE 23 Expression of Di-Alpha Hemoglobin in S. cerevisiae

Methods

Unless stated otherwise, all enzymes (restriction endonucleases, T4 DNAligase, T4 DNA polymerase, T4 polynucleotide kinase) were purchased fromNew England Biolabs, Pharmacia, BRL, Stratagene or Boerhinger Mannheim.Restriction enzymes and T4 DNA ligase were used with the bufferssupplied by the manufacturers.

Ethanol precipitation of nucleic acids was carried out by the additionof 0.5 volumes or 7.5M ammonium acetate and 2 volumes of 20:1ethanol-isopropanol. The pellet was collected by centrifugation at14,000×g for 15 minutes, washed twice with 80% ethanol, once with 95%ethanol and dried in vacuo. Phenol extractions were done by the additionof 50:49:1 mixture of phenol:chloroform:isoamyl alcohol. Phases wereseparated by centrifugation at 14,000×g and the aqueous phase collected.Plasmid DNA was purified from E. coli DH5α as described by Birnborn andDoly (Nucleic Acids Research 1979, 7:1513-1520). Electrophoreticanalysis of DNA was carried out in agarose gels using tris-acetateelectrophoresis buffer (Maniatis, et al. Molecular Cloning, Cold SpringsHarbor, N.Y., 1982). DNA was visualized by staining the gels with 0.5μg/ml ethidium bromide and exposing the gels to ultraviolet light. DNAfragments were purified from agarose gels using a kit purchased fromBIO-101 DNA fragments were purified from acrylamide gels by crushing theexcised gel fragment in 3.25M ammonium acetate and incubating overnightat 37° C. Gel fragments are removed by centrifugation (14,000×g, 15 min)and the DNA precipitated with ethanol. The precipitate is dissolved inTE (10 mM Tris HCl, pH 7.8, 1 mM Na₃ EDTA). Acrylamide gelelectrophoresis of DNA was done as described by Maniatis, et al.(Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1982).Bacteriological growth media and DNA transformation methods are asdescribed by R. W. Davis, et al. (Advanced Bacterial Genetics, ColdSpring Harbor Laboratory, New York, 1980). Media for the growth of S.cerevisiae has been described by F. Sherman et al. (Methods in YeastGenetics: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,1979). Transformation of S. cerevisiae with linear or circular DNA wascarried out as described by H. Ito, et al. (J. Bacteriology,153:163-168(1983)).

Removal of the PstI and SPeI sites from pGS4888.

The design of the synthetic linker for joining two α-globin chainsallows the inclusion of PstI and SpeI sites flanking a 30 bp sequencethat includes the junction of the two α-globin coding sequences. Becausewe anticipate testing several different linker sequences, these siteswill allow directional cloning of relatively short syntheticoligonucleotides encoding different linker sequences. Removal of thePstI and SpeI sites from the vector sequence is, therefore, necessary sothat the sites in the coding region are usable. One μg of the plasmidpGS4888 was digested with PstI and ethanol precipitated. The dry pelletwas resuspended in 50 μl of 33 mM Tris-acetate, pH 7.9, 66 mM potassiumacetate, 10 mM magnesium acetate, 0.5 mM DTT and 50 μM of each dNTP (T4polymerase buffer). Two units of T4 DNA polymerase were added and thereaction mixture incubated for 15 min at 37° C. Na₃ EDTA was added to12.5 mM and the reaction mixture heated to 65° C. for 15 min, phenolextracted and ethanol precipitated. The dry pellet was dissolved in 14μl of T4 DNA ligase buffer (BRL) and 1 μl (10 units) of DNA ligaseadded. The ligation mixture was incubated a 4° C. for 16 hr. A portionof the ligation reaction was used to transform E. coli DH5α andtransformants were selected on LB-ampicillin plates. Plasmid DNA wasprepared from 12 transformants. The DNA was analyzed by agarose gelelectrophoresis of PstI digests. Five transformants had lost the PstIsite and one of these was designated pGS1889. The SpeI site of thisplasmid was removed as described above after digestion of pGS1889 withSpeI. A plasmid was identified that had lost both the PstI and the SpeIsite and was designated pGS1989.

Strategy for Joining Two Copies of the α-globin cDNA

A fragment containing the 5'-363 bp of the α-globin coding region can beexcised from pGS4888 as an NcoI to ApaL1 fragment. A second fragmentcontaining nucleotides 109 through the 3' untranslated region can beremoved as a FokI to SalI fragment. These two fragments can then bejoined together to create a single translation unit encoding two tandemcopies the of α-chain cDNA by using a synthetic oligonucleotide adaptor(Table 8). The purification and assembly of these fragments is describedbelow.

The plasmid pGS4888 was sequentially digested with ApaL1 and NcoI and a.sup.˜ 365 bp fragment (α-chain 1) purified by acrylamide gelelectrophoresis. A second fragment of .sup.˜ 351 bp (α-chain 2) wasprepared by sequential digest with FokI and SalI followed by acrylamidegel electrophoresis. Four oligonucleotides were synthesized (Table 8)and assembled into a .sup.˜ 173 bp linker (RGGV) containing ApaL1 andFokI ends. Synthesis, purification and assembly of oligonucleotides wasas described previously. This adaptor encodes an amino acid bridgelinking the carboxy-terminus of α-chain1 with the amino-terminus ofα-chain2 as well as portions of the 3' end of α-chain1 and the 5' end ofα-chain2. The carboxy terminal arginine residue of α-chain1 is separatedfrom the amino terminal valine of α-chain2 in this construct by 2glycine residues. The "diglycine bridge" portion is flanked by HpaI,SpeI and PstI sites. These sites allow the substitution of a variety ofbridges by the use of .sup.˜ 30 bp adaptors to connect the two α-chains.The assembly of the two α-chain genes into an expression cassette wascarried out in a four part ligation as described below.

Plasmid pGS1989 was sequentially digested with NcoI and SalI and thelarge fragment containing the plasmid vector and pGALGAP was purified byagarose gel electrophoresis (gp1989). Fifty nanograms of gp1989 weremixed with 200 ng each of the gel purified ApaL1 -NcoI α-chain1 fragmentand the FokI-SalI α-chain2 fragment. A twenty-fold molar excess of thesynthetic ApaL1 -FokI adaptor was added (the 5'-ends of the adaptorsegment are not phosphorylated). The ligation reaction was carried outin a volume of 20 μl for 4 hr at 23° C. A portion of this reactionmixture was used to transform E. coli DH5α and ampicillin resistantcolonies were selected. Transformants were patched to nitrocellulosefilters and screened by hybridization with the ³² P-labelledoligonucleotide AL2as (Table 8). Five pMoles of oligo AL2-as wereincubated in 20 μl of a solution containing 2 units of T4 polynucleotidekinase, 50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂.6 mM DTT. 0.1 mMspermidine. 0.1 mM EDTA, and 10 pMoles of c³² P-ATP (7000 Ci/mMole) for2 hr at 37° C. Filters were processed as described by Maniatis, et al.and the hybridization was done at 37° C. in 5×SSC, 50% formamide, 100μg/ml yeast tRNA and 2% SDS for 16 hr. The filters were washed,sequentially, in 2×SSC and 1×SSC at 55° C. for 15 min (twice in each,all wash solutions contained 1% SDS). Dried filters were exposed toX-ray film and colonies giving a hybridization signal were used toprepare plasmid DNA. Plasmid DNA was analyzed by restriction enzymedigestion to identify plasmids that contained inserts of a sizeconsistent with two α-chains (NcoI-SalI digest) and that containedunique PstI, SpeI and HpaI sites. A plasmid identified in this mannerwas designated pGS2189 (FIG. 25).

An XhoI fragment from pS3888 containing the β-globin expression cassettewas purified by agarose gel electrophoresis. XhoI digested pGS2189 (50ng) was combined with 150 ng of the gel purified insert from pGS3888 in10 μl of ligation buffer containing 10 units of T4 DNA ligase. A portionof this mixture was used to transform E. coli DH5α and ampicillinresistant colonies were selected. Plasmid DNA was isolated and analyzedby digestion with XhoI, BamHI or NcoI. Several plasmids were identifiedthat produced restriction fragments of the expected sizes, all containedinserts in the orientation shown in FIG. 25. One of these was designatedpGS2989. Although this plasmid contains the linked α-globin genes and aβ-globin gene under the control of separate promoters, it is not capableof replication in S. cerevisiae. The entire expression cassette,containing the two genes (diα and β-globin), can be purified as a NotIfragment. Ten μg of pGS2989 was digested with PvuI and NotI and the NotIfragment gel purified. The digestion with PvuI was done to reduce thesize of the vector sequences which otherwise would comigrate with thedesired NotI fragment. Two hundred ng of the gel purified NotI fragmentwas combined with 50 ng of NotI digested pC1N in 10 μL of ligationbuffer containing 10 units of T4 DNA ligase. The reaction mixture wasincubated at 4° C. overnight and a portion was used to transform E. coliDH5α. Ampicillin resistant transformants were selected and plasmid DNAprepared. DNA was digested with NcoI-SalI, PstI or NotI to identifyplasmids with the diα, β-globin expression cassette and to determine theorientation of the inserted fragment. Several plasmids were identifiedthat contained the correct insert, all of which have the insertedfragment in the same orientation. One of these was designated pGS3089(FIG. 25). This plasmid was used to transform strains GSY112 and RSY334.

Expression and Purification

S. cerevisiae strains GSY112 pGS3089! and RSY334 pGs3089! were grown tosaturation in SD-uracil medium and diluted into 2 L of YPD medium (allcultures were incubated at 30° C.). Twenty-four hours after inoculation(of the YPD culture), galactose was added to 1% and the culturesincubated for another 24 hr. Carbon monoxide was bubbled through theculture and the cells collected by centrifugation. A 1:1 mixture(weight:vol) of cells and breakage buffer (10 mm sodium phosphate (pH7.2), 1 mM EDTA, 1 mM EGTA and 5 mM DTT) were disrupted in a"Bead-Beater" (Biospec Products, Bartlesville, Okla.). Debris wasremoved by centrifugation (10,000×g, 30 min). The soluble fraction wasadjusted to pH 6.0 with phosphoric acid and chromatographed on a columnof S-sepharose (fast flow) equilibrated in 10 mM sodium phosphate pH6.0. The loaded column was washed with 10 mM Tris-HCl, pH 6.7 andhemoglobin eluted by washing with 20 mM Tris-HCl, pH 7.5. A bright redband was collected and the pH adjusted to 8.0, by the addition of NaOH.This material was then chromatographed on Q-sepharose (fast flow)equilibrated in 20 mM Tris-HCl, pH 8.0. Hemoglobin was eluted with aNaCl gradient (0-0.4M). A final chromatography step was carried out onSephacryl S-200 equilibrated in 5 mM NaPO₄, pH 7.4, 0.1M NaCl. Each stepof the purification protocol was analyzed by SDS-polyacrylamide gelelectrophoresis (Laemmli, U.K., 1970, Nature 227:680-685) and stainingwith Commassie brilliant blue. The purified protein contains a band thatcomigrates with monomer β-globin and a band in the expected position forα-globin dimer. This material comigrates with human tetramerichemoglobin when analyzed by size exclusion chromatography (Progel TSKG3000 SWXL HPLC column). This protein is red and binds O₂ with a 50%binding affinity (P₅₀) of 8-10 Torr, indicating that it has incorporatedheme and is capable of reversible O₂ binding.

The purified protein was separated into α- and β-chains by reverse phaseHPLC and the sequence of the 10 amino-terminal residues of each chainwere determined. The sequence matched that of bona fide humanhemoglobin, indicating that the initiating methionine had beenefficiently removed from both the α-globin dimer and the β-globin dimer.

EXAMPLE 24 Construction of Low Affinity. Genetically Cross-Linked,Hemoglobin Mutants and Expression in Yeast

24.1 Construction of vector for site directed mutagenesis

The XhoI fragment containing the β-globin gene and its GALGAP promoterwas isolated from pGS2989 by preparative agarose gelelectrophoresis andligated with XhoI digested Phagescript (RF-form, obtained fromStratagene, Inc.). E. coli XL1-Blue was transformed with the DNAligation mixture and phage containing inserts were identified (whiteplaques on medium containing XGAL). Single plaques were isolated and DNAwas prepared and analyzed by digestion with XhoI and agarose gelelectrophoresis. This construct was designated M13β-globin.

24.2 Preparation of single stranded template

A saturated culture of E. coli XL1-Blue (available commercially fromStratagene, 11099 North Torrey Pines Road, LaJolla, Calif. 92037) (200μl) was used to inoculate 4 ml of 2× YT broth. This culture wasincubated for 2 hr. at 37° C. and then infected with a singleM13β-globin phage plaque and the incubation continued for 6-8 hrs. Cellswere removed by centrifugation and discarded. Phage were precipitatedfrom 1.6 ml of clarified medium by the addition of 320 μl of cold 30%PEG 8000 in 3.5M ammonium acetate followed by incubation on ice for 30min. Phage were collected by centrifugation and resuspended in 0.1 ml of10 mM Tris-HCl pH 8.0, 1 mM EDTA (TE). DNA was isolated by extractingtwice with phenol/cholorform. DNA (contained in the aqueous phase) wasprecipitated by the addition of NaCl to 0.5M and two volumes of 95%ethanol. The DNA pellet is dissolved in 20 μl of water.

24.3 In vitro mutagenesis reactions

Two hundred ng of template DNA are mixed with a twenty-fold molar excessof the appropriate, phosphorylated mutagenic oligonucleotide in 10 μl of20 mM Tris-HCl, pH 7.4, 2 mM MgCl₂, 50 mM NaCl and heated to 70° C. for5 minutes. The annealing reaction is allowed to slowly cool (40 min) to40° C. and then to 30° C. (15 min). After the last annealing step themixture is transferred to ice for 5 min. To this mixture 1 μl ofsynthesis buffer (4 mM each dNTP, 7.5 mM ATP, 175 mM Tris-HCl pH 7.4,37.5 mM MgCl₂ 215 mM DTT), 0.5 μl of T4 gene32 protein (2 μg/μl), 1 μlof T4 DNA ligase (3 units), and 1 ml of T4 DNA polymerase (1 unit) areadded. The mixture is incubated at 37° C. for 90 min (following 5 min atroom temperature). The reaction is stopped by the addition of 90 ml of0.1M Tris-HCl (pH 8.0) and 0.1M EDTA. Approximately 0.1 μl of thereaction mix was used to transfect E. coli XL1-Blue cells (Cells wereprepared for transformation by the method of D. Hanahan, 1983. J. Mol.Biol. 166:557).

24.4 Screening for phage containing the mutant gene

Approximately 50-100 plaques were picked to fresh plates seeded with theappropriate host strain (XL1-Blue) in ordered arrays. After incubationfor 6-8 hrs. at 37° C. the plates were overlaid with nitrocellulosefilters and prepared for hybridization essentially as described inDavis, R. W. et al. (Advanced Bacterial Genetics: A. Manual of GeneticEngineering. Cold Spring Harbor Laboratory, New York 1980). The choiceof hybridization temperature with the mutagenic oligonucleotide wasdetermined on the basis of the nucleotide composition of theoligonucleotide. The oligonucleotide was labelled with c³² P-ATP andpolynucleotide kinase (T. Maniatis et al. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory, 1982). Hybridizationswere done at 2°-5° C. below the calculated Tm for the correct match in6×SSC, 2% SDS, 100 μg/ml yeast tRNA using 10⁵ cpm/ml of the labelledoligonucleotide for >6 hr. The Tm was calculated using the formula: 4°C. for every GC pair +2° C. for each AT pair assuming an Na+concentration of 1M. Filters were washed at the same temperature as thehybridization in 5×SSC, 2% SDS and exposed to XRay film. Plaques givingpositive hybridization signals were used to prepare single stranded DNA,as described above. The single-stranded DNA was used as template forsequencing reactions (Sanger, F. and Coulson, A. R. 1975, J. Mol. Biol.94:441) to confirm that the mutant sequence was indeed present.

Oligonucleotides used for mutagenesis.

βN108K-"Presbyterian" 5'-AGGCTCCTGGGCAAGGTGCTGGTCTGT-3'

βE90K-"Agenogi" 5'-GCCACACTGAGTAAGCTGCACTGTGAC-3'

βV67I-(No Alias) 5'-CATGGCAAGAAAATCCTCGGTGCCTTT-3'

βN102T-"Kansas" 5'-GTGGATCCTGAGACTTTCAGGCTCCTG-3'

24.5 Cloning into yeast expression vectors

Phage RF DNA was prepared from phage-infected cells that had beenconfirmed to have the mutant sequence and the XhoI fragment containingthe altered β-globin gene was purified by agarese gel electrophoresis.This fragment was cloned into a derivative of pGS3089 that had beenaltered to change the di-α-crosslink from the diGly configuration to asingle glycine bridge, and from which the β-globin gene had beendeleted, and designated pGS3889RGVdesβ (FIG. 26). This created a uniqueXhoI site into which altered β-globin expression cassettes could beinserted, allowing coexpression with the α-globin dimer. The expressionplasmids generated in this way (pGS3889 for "Presbyterian", pGS5189 for"Agenogi", pGS5689 for "Kansas" and pGS4989 for "βV67I", all identicalto pGS3089 except for mutation in specified codon) were used totransform S. cerevisiae strains GSY112 and RSY334.

24.6 Characteristics of a genetically crosslinked low affinity mutantprotein expressed in yeast

Cells bearing the plasmid pGS3889 (single gly bridge, βN108K alias"Presbyterian") were grown and hemoglobin purified as previouslydescribed. This material when analyzed for functionality wassubstantially "right-shifted" compared to the crosslinked protein with awild type β-chain (P₅₀ =23-25 for the mutant, with N=2.5).

EXAMPLE 25 Effect of Choice of Strain and Induction Temperature onExpression of Di-Alpha Hemoglobin in E. coli

Table 100 contains comparisons of di-alpha/beta fermentations. It alsoprovides comparison of temperatures of induction. The column labeled "mgdi-A+B" is total mg di-alpha and beta polypeptides per fermentation. Theadjacent column "mg/OD-L" simply expresses the first column number on acell density basis. The two columns labeled RHGB present total and celldensity-corrected output of functional recombinant hemoglobin. The lastcolumn shows that in terms of final functional hemoglobin recovery,strain JM109 is preferable. Without binding ourselves to any theory, webelieve that this difference has to do with the proteases expressed indifferent strains. It is interesting to note that of the two best JM109runs, one induction was at 30° and one at 37° with roughly equivalentamounts of final functional Hb produced.

EXAMPLE 26 Construction of Genetically Cross-Linked α-Globin DimersConnected By a Single Glycine or Proline Residue

The following synthetic adaptors for altering the dia-globin bridge weresynthesized and purified as described in previous sections.

    __________________________________________________________________________       T  S  K  Y  R  P  V  L  S  P   A                                           5'-A                                                                             ACT                                                                              AGT                                                                              AAG                                                                              TAC                                                                              AGA                                                                              CCT                                                                              GTT                                                                              TTG                                                                              TCT                                                                              CCT GCA-3                                                                             RPV                                     3'-T                                                                             TGA                                                                              TCA                                                                              TTC                                                                              ATG                                                                              TCT                                                                              GGA                                                                              CAA                                                                              AAC                                                                              AGA                                                                              GG-5'                                              HpaI                       PstI                                               T  S  K  Y  R  G  V  L  S  P   A                                           5'-A                                                                             ACT                                                                              AGT                                                                              AAG                                                                              TAC                                                                              AGA                                                                              GGT                                                                              GTT                                                                              TTG                                                                              TCT                                                                              CCT GCA-3'                                                                            RGV                                     3'-T                                                                             TGA                                                                              TCA                                                                              TTC                                                                              ATG                                                                              TCT                                                                              CCA                                                                              CAA                                                                              AAC                                                                              AGA                                                                              GG-5'                                              HpaI                       PstI                                            __________________________________________________________________________

Complementary pairs of RPV and RGV oligonucleotides were combined (2.4μg of each oligonucleotide) in 0.05 ml of water. The two pairs ofadaptors were precipitated (separately) by the addition of 2. μl of 4MNaCl and 0.158 mL of 100% ethanol followed by centrifugation. Thepellets were washed with 80% and 100% ethanol and dried. The adaptorswere dissolved in 24 μl of TE.

26.1 Cloning of the RGV and RPV adaptors

The plasmid of pGS2989 was sequentially digested with the enzymes HpaIand PstI and the vector was purified after agarose gel electrophoresis.The digested plasmid was then ligated with a 10-fold molar excess ofeither the RGV or RPV adaptor (these adaptors were not phosphorylated)as previously described. A portion of the ligation mixture was used totransform E. coli DH5. Transformants were selected on LB platescontaining ampicillin. Clones containing the new adaptor were identifiedby colony filter hybridization. The hybridization probes were either theRPV or RGV upper strands (as shown above). These proves were labelledwith c32P-ATP and T4 polynucleotide kinase. Filters were hybridized withthe appropriate probes (10⁵ cpm/ml) at 37° C. in a solution containing6×SSC. 50% formamide, 2% SDS and 150 μg/ml yeast tRNA. Filters wereincubated for >12 hrs. and washed 4 times with 2×SSC, 2% SDS (250 ml, 20min each) and exposed to X-ray film. Colonies that producedautoradiographic signals were used to prepare plasmid DNA which wassequenced using the following primer: 5'-AAGCTTCAGCACCGTATTCA-3' (α²seql). This primer was specifically designed to minimize homology withthe corresponding sequence in the α1 subunit of the dimer and tomaximize homology with sequences near the 5' end of the α2 subunit ofthe diα dimer. This allows sequence to be determined reading from the αregion through the sequence that bridges the two α-domains without abackground sequence reading from a similar sequence near the 5'-end ofthe α1-domain. The plasmids constructed in this way were designatedpGS2989RPV(single proline crosslink) or pGS2989RGV (single glycinecrosslink).

26.2

Cloning into yeast expression Plasmids

The NotI fragments from either pGS2989RPV or pGS2989RGV that contain thehemoglobin expression cassette were purified by agarose gelelectrophoresis and subcloned into pC1N that had been digested withNotI. Plasmid DNA from ampicillin resistant transformants was isolatedand analyzed by digestion with NotI and agarose gel electrophoresis.These plasmids were designated pGS3089RPV or pGS3089RGV. A second set ofplasmids was generated from these to facilitate the substitution ofdifferent β-chain mutants. These plasmids were generated by digestionwith XhoI and religation under dilute conditions (<1 μg/ml). This favorsdeletion of the β-chain expression cassette. Plasmid DNA was isolatedfrom ampicillin resistant transformants and the structures confirmed bydigestion with NotI and XhoI (these plasmids have been designatedpGS3089RGV-desβ, see FIG. 26, and pGS3089RPV-desβ).

EXAMPLE 27 Comparison of Renal Toxicity of Di-Alpha Hemoglobin andDes-Val Hemoglobin

Safety studies were conducted in both rats and dogs. Two rats receivednon-stabilized rHb and two rats received stabilized rHb (a di-alpha Hbwith a di-glycine linker between the alpha globin domains) The followingserum chemistry parameters were evaluated:

CK creatinine kinase

ALT alanine aminotransferase

AST aspartate aminotransferase

BUN blood urea nitrogen.

A substantial increase in these parameters, relative to baseline, 24hours after administration, is indicative of renal toxicity: Thefollowing values were obtained:

    ______________________________________                                                         Non-                                                                          Stabilized                                                                             Stabilized                                          TEST             rHb      rHb                                                 ______________________________________                                        BUN baseline     19.5     17.5                                                BUN 24 hours     109.5    26.5                                                CK baseline      0.45     0.5                                                 CK 24 hours      1.35     0.5                                                 AST(SGOT) baseline                                                                             155.5    212.5                                               AST(SGOT) 24 hours                                                                             3444.0   942.0                                               ALT(SGPT) baseline                                                                             62.0     80.5                                                ALT(SGPT) 24 hours                                                                             958.5    192.5                                               ______________________________________                                    

It is noted that the increase for the stabilized rHb is much lesspronounced.

Dogs were first stressed by withdrawing approximately 30% of their bloodvolume, allowing them to stabilize for 30 mins, and then resuscitatingwith either rHb1.1, saline, human serum albumin or autologous blood.

Histopathology

No macroscopic or microscopic lesions were observed in organs (i.e.,kidneys) at the end of the study that could be directly attributed tothe administration of recombinant, stabilized hemoglobin.

Serum Chemistry

Serum chemistry data for dogs treated with rHb1.1 were evaluated forstudy day 2 (22-24 hours after hypovolemic stress) for CK (creatineKinase), ALT (Alanine Aminotransferase), AST (AspartateAminotransferase) and Urea (Blood Urea Nitrogen, BUN). The AST and CKwere notably increased in the rHb1.1 treated animals at day 2, however,these values returned to pretreatment levels by study day 4.

Urine Biochemistry

Urinalysis was not affected by the administration of rHb1.1. Other thana very slight decrease in NAG activity in all females (in alltreatments) on study day 2, no notable findings were observed.

In summary, the magnitude and time course of cardiovascular andhemodynamic effects observed in severely hemorrhagic dogs resuscitatedwith recombinant di-alpha hemoglobin were nearly identical to thoseobserved in dogs resuscitated with autologous blood. This studydemonstrates that recombinant hemoglobin does not cause adversecardiovascular, or hemodynamic effects when compared to HSA orautologous blood resuscitation in severely stressed dogs. Moreover, 30%blood volume resuscitation with recombinant di-alpha hemoglobin (1.1g/kg) in hypovolemic dogs produced only limited, reversible effects,including transient neutropenia and elevation of blood plasma enzymes ofskeletal muscle origin. No major organ systems were affected, nohemoglobin was found in the urine of recombinant hemoglobin treateddogs, and no histopathological changes were observed.

EXAMPLE 28 Construction of di-α globin mono-cysteine (A71C, D75C, orS81C) mutant expression vector

28.1 Subcloning of the α gene into phagescript

The desfxα pGem (pDLII-83α) vector was cut with EcoR1 and Pst1endonucleases and ligated into EcoR1/Pst1 digested phagescript (obtainedfrom Stratagene). E. coli strain DH5α was transformed with the ligationmixture and cells were plated on 2×YT plates overlaid with 3 ml top agarcontaining 10 μl 100 mM IPTG, 25 μl 2% X-Gal in DMSO and 150 μl XL-1cells (Stratagene). Clear plaques were picked and grown at 37° C. in2×YT containing XL-1 cells. Double stranded DNA was isolated from thecultures and checked for the presence of the 500 bp α gene byrestriction analysis and agarose gel electrophoresis. Single strandedDNA was isolated from one of the desfxα phagescript transformants (namedf 191). The single stranded DNA was sequenced to confirm the presence ofthe desfxα gene in the phagescript.

28.2 Mutagenic Oligonucleotides

Three mutagenic oligonucleotides were used in three separate mutagenicreactions. The sequences of the oligonucleotides were as follows (mutantcodon is underlined):

Nigeria mutation: αS81C

5' CCG AAC GCG TTG TGC GCT CTG TCT GAT 3' (SEQ ID NO:5) αD75C

5' GGT GCT CAC GTT GAT TGC ATG CCG AAC GCG 3' (SEQ ID NO:6) αA71C

5' CTG AAC GCT GTT TGC CAC GTT GAT GAT 3'

28.3 Kinase reaction conditions for mutagenic oligonucleotides A71C.D75C and S81C.

1 μl oligonucleotide (approx. 300 pmol)

2 μl 10× kinase buffer containing 10 mM ATP

0.5 μl T4 polynucleotide kinase (10 U/μl, New England Biolabs)

15.5 μl H₂ O

1 μl 10 mM spermidine

Reactions were incubated for 1 hr. at 37° C., then 80 μl H₂ O was addedand the reaction was terminated by heat inactivation at 65° C. for 10min.

28.4 Mutagenesis Reaction

1 μl f 191 ss DNA (0.5 pmol)

3 μl kinased oligonucleotide (either A71C, D75C or S81C approx. 45 pmol)

2 μl 10× annealing buffer (Promega)

14 μl H₂ O

The no primer control contained:

1 μl f 191 ss DNA

2 μl 10× annealing buffer

17 μl H₂ O

Reactions were heated to 65° C., cooled slowly to 35° C. (approx. 70min), and put on ice for 5 min. The following reagents were added andthe reactions were incubated at 37° C. for 90 min.

3 μl 10× synthesis buffer (Promega)

1 μl T4 gene 32 protein (0.5 μg/μl, Biorad)

1 μl T4 DNA polymerase (3 U/μl, NEB)

0.5 μl T4 DNA ligase (10 U/μl, NEB)

5 μl H₂ O

200 μl 71-18 mut S competent cells (made according to Promega AlteredSites procedure) were transformed with 10 μl of each mutagenesisreaction, put on ice for 30 min and heat shocked for 2 min at 42° C. Thetransformation mixture was added to 3 ml 2×YT media and grown at 37° C.(with shaking) for 5.5 hr. After incubation, 1 ml of each of the threecultures was removed, centrifuged and 800 μl was stored in a fresh tubeat 4° C. as the stock solution of mutant phage.

28.5 Screening for mutants of D75C

100 μl of a 10⁻⁵ dilution of the D75C mutant phage stock was plated on153 mm 2×YT plates overlain with top agar containing 0.5 ml XL-1 cells.Plates were incubated at 37° C. for approx. 5 hrs. Duplicatenitrocellulose filters were lifted off each plate and the plaques werelysed in 6 ml 0.5M NaOH/1.5M NaCl, neutralized in 10 ml 1M Tris-HCl pH8.0/1.5M NaCl and washed in 500 ml 6×SSC. The filters were air dried andbaked at 75° C. for 45 min. The filters were then boiled briefly in 1%SDS prior to prehybridization. Filters were prehybridized in 20 mlsolution for 4 hr at 68° C. The prehybridization solution was asfollows:

5×SSC (20× SSC prepared according to recipe in Maniatis).

0.1% (w/v) N-lauroylsarcosine

0.02% (w/v) SDS

0.5% blocking reagent (Genius Kit, Boehringer Mannheim)

The D75C oligonucleotide was labelled with y 32!PATP as follows:

1 μl oligonucleotide (80 pmol)

10 μl 10× kinase buffer

1 μl γ.sup. 32! P ATP (10 μC/μl. Specific activity >3000 Ci/mmol).

87 μl H₂ O

1 μl kinase (10 U/μl, NEB)

The reaction was incubated for 5 hrs. at 37° C. Unincorporated ATP wasremoved by centrifugation through a Biospin 30 column (Biorad). Theentire probe (17,000 cpm/μl) was added to the prehybridization mixtureand the filters were hybridized overnight at 46° C. along with a noprimer control filter. The following day, filters were washed for 10min. at room temperature (RT) in 6×SSC and exposed overnight at -70° C.on Kodak X-Omat film. Filters were washed in 6×SSC at 57° C. for 10 min,dried and exposed overnight, then washed in 6×SSC/0.1% SDS at 67° C. for10 min, and dried and re-exposed overnight. The final was was in6×SSC/0.1% SDS at 70° C. for 10 min and the filters were again dried andexposed overnight.

Ten plaques were picked which hybridized differentially to the mutantoligonucleotide (compared to the no primer control plaques). The plaqueswere placed in 5 ml 2×YT media containing 0.25 ml XL-1 cells. Thecultures were incubated with shaking at 37° C. for 7.5 hr. 1 ml of eachculture was removed, centrifuged 5 min,. placed in a fresh tube andstored at 4° C. for subsequent sequencing and plaque purification.

28.6 Screening for mutants of A71C and S81C

1 μl of a 10⁻³ dilution of the A71C stock phage mutagenesis reaction and20 μl of a 10⁻⁵ dilution of the S81C mutagenesis reaction were plated onfour separate 82 mm 2×YT/tet(10 mg/ml) plates overlaid with 3 ml topagar and 100 μl XL-1 cells. A no primer control was also plated asabove. The plates were incubated at 37° C. for 5 hr; plaques were liftedfrom each plate onto nitrocellulose filters and the filters driedovernight at room temperature. The following day, the plaques were lysedwith 0.5M NaOH/1.5M NaCl for 3 min, neutralized in 1M Tris-HCl pH7.0/1.5M NaCl for 3 min and washed in 6×SSC for 5 min. Filters were airdried then baked at 75° C. for 1 hr. The filters were boiled briefly in1.5% SDS prior to prehybridization at 60° C. for 6 hr. in 10 mlprehybridization solution as described above.

28.7 Labelling of A71C and S81C oligonucleotides using digoxigenin

(All reagents supplied by Boehringer Mannheim)

2 μl A71C (100 pmol) or 1 μl S81C (110 pmol)

10 μl terminal transferase buffer

5 μl 25 mM CoCl₂

1 μl 1 mM dUTP-digoxigenin

30 μl-31 μl H₂ O (A71C and S81C reactions respectively)

1 μl terminal transferase (25 U/μl)

Reactions were incubated at 37° C. for 3 hr. followed by 6 hr. at RT.Digoxigenin-labelled A71C and S81C probes (20 μl) were added to theappropriate filters in 10 ml prehybridization solution along with a noprimer control filter. The filters were hybridized overnight at 47° C.

28.8 Filter Washes and Development

All filters were initially washed in 6×SSC/0.1% SDS for 15 min at 30°C., then for 15 min at 42° C.

Each of the four filters which had been probed with either the labelledA71C or S81C oligonucleotides were then separated and washed atincreasingly higher temperatures along with a no primer control filter.One each of the A71C and S81C filters were placed in plastic bagscontaining 10 ml of 6×SSC/0.1% SDS and washed for 10 min at one of threetemperatures, i.e., 50° C., 60° C. or 65° C. After the high temperaturewashes, each set of filters were developed according to the Genius Kitprotocol.

Initially, bags containing the filters were filled with 10 ml of 100 mMTris-HCl, pH 7.5/150 mM NaCl (buffer A) and incubated for 15 min. Thebuffer was removed and replaced with 10 ml buffer A containing 0.5%blocking reagent and incubated a further 15 min at RT without shaking.Anti-digoxigenin antibody (2 μl) was added directly to each bag andincubated with for 30 min at RT. The filters were then removed fromtheir respective bags and washed altogether in 100 ml buffer A/0.05%blocking reagent for 15 min at RT, followed by a 15 min wash in buffer Aalone at RT. The final wash was 100 ml 100 mM Tris-HCl, pH 9.5/100 mMNaCl/50 mM MgCl₂ (buffer B) for 5 min at RT. Each set of filters from agiven temperature was placed in a separate bag along with 5 ml of colordevelopment solution (5 ml buffer B containing 22.5 μl 75 mg/ml NBT/15μl 50 mg/ml X-phosphate). The filters were incubated for 30 min in thedark at RT. After 30 min, the filters were removed from the developmentsolution, washed for 5 min in 100 ml 10×TE and 5 min in 100 ml 1× TE,both at RT. Filters were dried at RT.

Using the results from the Genius Kit screening procedure, 10 plaqueswhich differentially hybridized to the labelled oligonucleotides A71C orS81C were picked and placed in 3 ml 2×YT media containing 0.25 ml XL-1cells and incubated for 7.5 hr. at 37° C. with shaking. 1 ml of eachculture was removed, centrifuged 5 min, placed in a fresh tube andstored at 4° C. for subsequent sequencing and plaque purification.

28.9 Confirmation of mutations by sequencing

Single stranded DNA was isolated from 800 μl mutant phage stocksupernatant and sequenced using the Sequenase kit (USB) with the α 179oligonucleotide as the primer. The α 179 aligonucleotide is an 18-merhomolog cys to a region about 100 bps upstream of the mutation site.)Sequencing confirmed the presence of the αA71C, αD75C and a S81Cmutations.

Phage stock was plaque purified by plating 10 μl of 10⁻⁸ and 10⁻¹⁰dilutions on 2×YT/tet (10 mg/ml) plates overlaid with 3 ml top agarcontaining 200 μl XL-1 cells. After 7 hrs incubation at 37° C., a singleisolated plaque from each mutant plate was picked and used to inoculate90 ml 2×YT/tet (10 mg/ml) media containing 10 ml XL-1 cells. Cultureswere grown overnight at 37° C. with shaking. 1 ml of each mutant phageculture was removed, centrifuged and the supernatant was frozen at -80°C. as the respective purified mutant phage stock. Double stranded DNAwas prepared from the remaining culture for use in the subsequentsubcloning steps into the final expression vector 1.1E4.

28.10 Subcloning of the α cys mutants into 1.1E4

Construction of the di-α gene with each of the three cysteine mutationsin either the N-terminal or C-terminal domain of the di-α proteinrequired three subcloning steps:

1) Transfer of the cys mutant α gene from phagescript as an Eag1-Pst1fragment into the Eag1-Pst1 digested desval α pGem vector (pDL II-91f,see #10 table 200). This step provided the mutant a gene with thecorrect 5' terminus.

2) A mutant di-α gene with each of the cys mutations in the 3' a genewas constructed by inserting the Eag1 DNA fragment from di-α pGem (seepGem di-alpha, #5a, table 200) into the Eag1 site of the relevant cysmutant desval α pGem plasmid. The mutant diα gene with the cys mutationin the 5' α gene was constructed by inserting the BstB1 DNA fragmentfrom diα pGem into the BstB1 site of the cys mutant desval α pGemplasmid.

3) Finally each of the mutant dia genes were cloned into the 1.1E4expression vector as a Sma1-Pst1 fragment.

Transformations into DH5α at each step in the subcloning procedure werecarried out as described in the methods of subcloning of the β G83Cmutation into 1.1E4 (see below). The presence of the relevant cysmutation in the correct α gene was confirmed by sequencing at each stagein the subcloning procedure. Each of the diα cys mutants in 1.1E4 weretransformed into E. coli strain 127, grown in TB complete media andinduced with IPTG. Expression of the diα and β proteins was confirmed bySDS-PAGE and Western blot analysis.

EXAMPLE 29 Hypothetical protocol for the oxidation of two SGE1.1mono-cys's to form a pseudo-octamer

The surface cysteine mutants (MW=64 kDa) can be oxidized to thedisulfide-linked dimer under oxidative conditions. This can beaccomplished by stirring a concentrated solution of the expressedprotein at pH 8 under pure oxygen at 4° C. or room temperature in thedark. Trace levels of transition metal ions such as CU⁺² may be added tolevel below 1 μM to catalyze the oxidation (1). Formation of the 128 kDaoctamer can be monitored by gel filtration. Saturation of the solutionwith oxygen at elevated pH should minimize autooxidation of recombinanthemoglobin.

An alternative procedure, which may be the preferred method ofcatalyzing this reaction, involves the use of redox buffers such asreduced and oxidized glutathione, or reduced and oxidized dithiothreitol(2). This catalysis of the reaction through disulfide interchange may benecessary to control trace transition metal catalysis (3). An second,similar approach involves conversion of the surface cysteines in the 65kDa species to sulfonates before purification (to avoid 128 kDa speciesformation during purification), followed by conversion to thedisulfide-linked 128 kDa species with reduced glutathione (2).

(1) Freedman, R. B. and Hillson, D. A. (1980) "Formation of DisulfideBonds"IN: The Enzymology of Post Translational Modification of Proteins,Vol. 1, p. 157 ff. (Academic Press).

(2) DiMarchi, R., et al. (1988) Chemical synthesis of human epidermalgrowth factor (EGF) and human type a transforming growth factor (TGFa)IN: Peptides: Chemistry and Biology (G. R. Marshall, ed.) pp. 202-203(Leiden:ESCOM).

(3) Creighton, T. E. (1978) Experimental studies of protein folding andunfolding. Prog. Biophys. Molec. Biol. 33:231-297

EXAMPLE 30 Characterization of Di-Alpha Hemoglobin

We constructed an E. coli expression vector that contains two copies ofthe α globin gene fused in tandem by a single codon encoding a glycineresidue; this created a fusion junction with the sequenceArg(141α1)-Gly-Val(1α2). In this construct a single operon encoding adiα-globin and a β-globin chain is transcribed from a single TACpromoter. Hoffman, S. J., Looker, D. L., Roehrich, J. M., Cozart, P.,Durfee, S., Stetler, G., Proc. Nat. Acad. Sci. (U.S.A.) 87, 8521-25(1990). To decrease the oxygen affinity of this engineered Hb we haveintroduced an additional Asn-108β→Lys mutation. In its naturallyoccurring form (Hb Presbyterian) this substitution reduced the oxygenaffinity and slightly increased the Bohr effect. Moo-Penn, W., Wolff,J., Simon, G., Vacek, J., Jue, H., Johnson, M., FEBS Lett. 92, 56-57(1978). Upon induction of the Hb operon with IPTG, E. coli cellscarrying a plasmid containing the fused α globin gene produce twopolypeptides corresponding to β globin and fused diα globin.

To fully assess the separate effects of the α-globin fusion and theAsn-1908β→Lys mutation, we also constructed vectors to express fourrelated proteins: (1) A mutant form of the tetramer that contains Val toMet changes at the amino termini of both α and β chains and theAsn-108β→Lys mutation (rHb1.0); (2) pseudotetrameric diα-haemoglobinwith Asn-108β>Lys and amino-termini modified as described above(rHb1.1); (3) a pseudotetramer without the Asn-108β→Lys (rHb0.1); and(4) tetramer with neither the α fusion nor the β globin mutation(rHb0.0). The four forms of recombinant hemoglobin described above werepurified from E. coli and analyzed for functionality. The table belowsummarizes these results and compares them to adult human nativehaemoglobin (Hb A) purified blood. For analytical methods, see Hoffman,S. J., Looker, D. L., Roehrich, J. M., Cozart, P., Durfee, S., Stetler,G., Proc. Nat. Acad. Sci. (U.S.A.) 87, 8521-25 (1990). Wagenbach, M.,O'Rourke, K., Vitez, L., Wieczorek, A., Hoffman, S., Durfee, S.,Tedesco., J. Stetler, G. Biotechnology, 9, 57-61 (1991). The Bohrcoefficient is the slope of the line generated by plotting log P₅₀ as afunction of increasing pH (pH 6.8-7.8). Oxygen equilibrium binding datawere collected at 20° C. in 50 mM HEPES beffer containing 100 mM NaCl.Also shown are the amino acids present at the amino termini of the α andβ-chains, at position 108 of the β-chain and the structure of theα-chain.

The Asn-108β→Lys mutation causes a substantial decrease in oxygenaffinity of both the pseudotetramer and the tetramer when compared towild type human haemoglobin or rHb0.0. The Asn-108β→Lys mutationapparently also causes an increase in the Bohr effect which isconsistent with the initial characterization of human Hb Presbyterian.Fusion of the two α globin subunits causes a substantial increase inoxygen affinity and a slight decrease in cooperativity. Finally, thevaline to methionine alteration at the amino termini of the α and βsubunits results in a decrease in the oxygen affinity that is apparentlycompletely compensated for by the fusion of the two α-globin subunits.

    ______________________________________                                                 WT     1.1    1.0       0.1  0.0                                     ______________________________________                                        Bohr Coefficient                                                                         -0.47    -0.32  -0.35   -0.25                                                                              -0.27                                 p50 (torr) (20° C.)                                                               4.5      17.2   19.8    4.5  7.3                                   Hill Coeff (n max)                                                                       2.90     2.35   2.53    2.04 2.59                                  α-chain                                                                            WT       diα                                                                            WT      diα                                                                          WT                                    α-chain (NH3)                                                                      VL       ML     ML      ML   ML                                    β-chain (β108)                                                                 N        K      K       N    N                                     β-chain (NH3)                                                                       VH       MH     MH      MH   MH                                    ______________________________________                                    

In order to study the effect of these modifications on the structure ofHb we crystallized rHb1.1 in the deoxy form and solved the structure byX-ray crystallography. FIG. 32. shows a symmetry averaged differenceFourier map of rHb1.1 minus deoxy Hb A in the C-terminal region of one αsubunit. The peptide linkage between the two a subunits breaks themolecular dyad symmetry of the Hb tetramer. In deoxyhemoglobin crystalswith space group P21, the molecular two-fold axis is not acrystallographic two-fold axis, so that the electron density mapsoutline the structure of the symmetry-related αβ-dimers separately. InFIG. 32 these are superimposed. The filled bonds and single broken bondsrepresent approximate coordinates of the two symmetry-relatedconfigurations of the rHb1.1 α-chain termini: α1-Gly-α2 (open bonds) andα2-Gly-α1 (broken bonds). α1 and α2 refer to two α subunits related by anon-crystallographic dyad axis in each asymmetric unit of p21deoxyhemoglobin crystals, but not the order in the tandemly duplicatedpolypeptide. The similarity of the unaveraged difference map in the tworegions near Na1α1/HC3α2 and NA1α2/HC3α1 indicates that the twoconfigurations are present at nearly equal occupancies. Positivedensities bridging the N- and C-termini indicate that the glycineresidue links the C-terminus of one α chain to the N-terminus of thesymmetry related α subunits. The negative and positive densities arounda few residues near both N- and C-termini indicate that fusion of thetwo α subunits slightly constrains the conformation of both termini. TheAsn-108β→Lys 5 mg/ml mutation causes substantial structural changesincluding a shift of the B and G helices shown by paired positive andnegative densities along these two helices (not illustrated) but thestructural basis of the reduced affinity is not clear.

To test whether or not the fusion of the α subunits extends theintravascular half-life of hemoglobin, we prepared radioactivelylabelled rHb1.1 and rHb1.0 for in vivo studies in rats. As shown in FIG.33, rHb1.1 containing fused α subunits shows a significant increase inintravascular half-life compared to its non-fused homologue. Morecomprehensive toxicology and pharmacokinetic studies in rats and dogsindicate that renal toxicity associated with tetrameric hemoglobin hasbeen eliminated by the fusion of the two α-chains of rHb1.1. Theseresults are consistent with those reported for chemically crosslinkedhemoglobins. Bucci, E., Razynska, A., Urbaitis, B., Fronticelli, C., J.Biol. Chem. 264, 6191-95 (1989); Manning, L. R., et al., Proc. Natl.Acad. Sci (U.S.A.) 88, 3329-33 (1991). However, in contrast tochemically cross-linked hemoglobins, large amounts of genetically fuseddiα-haemoglobin can be produced by simple microbial fermentations andpurified without the additional steps or chemicals required to producechemically cross-linked haemoglobins. Our novel engineered Hb thereforeprovides a most promising way of producing a safe blood substitute.

EXAMPLE 31 Hypothetical Protocol for the Construction of HemoglobinMolecules Stabilized against dimer Formation by fusion across the alpha1- beta 2 or alpha 2- beta 1 dimer interface region

The currently employed inter-dimer di-alpha fusion between the Cterminus of one alpha subunit and the N terminus of the adjacent alphasubunit, represents a successful protein engineering approach tostabilizing hemoglobin against dimer formation. In this case, use wasmade of the fortunate juxtaposition of the two termini which originatefrom different dimers. One might also make a di-beta polypeptide, as hasbeen described, or a hemoglobin with both di-alpha and di-betapolypeptide, as has been described, or a hemoglobin with both di-alphaand di-beta linked subunits. Alternatively, one can envision other typesof fusion in which the alpha subunit of one alpha/beta dimer is fused tothe beta subunit of the other dimer (FIG. 1). In this, two individual,linked polypeptides would dimerize to form the psuedo-tetramerichemoglobin. This approach is based on the fact that dimerizationinvolves specific, identical pairs of subunits, generally referred to asα1β1 and α2β2.

As an example of this alternative fusion approach, the alpha subunit Cterminal residue (Arg 141) of dimer 1 could be fused, either directly orwith an intervening fusion sequence, to the N-terminal amino acid of thebeta subunit C helix (Tyr 35) of dimer 2. This would create a new Cterminal residue at the end of the beta B helix (Val 34) and would leavea "free" piece of polypeptide comprised of the beta A and B helices(residues 1 to 34 inclusive). These alterations would give rise to aprotein comprised of alpha subunit helices A through H fused to betasubunit helices C through H. The polypeptide composed of the betasubunit A and B helices would be covalently attached to the protein byintroducing a new helix into the molecule. The helix would be designedto span the distance between the beta C terminus (His 146) and theoriginal beta N terminus of helix A (Val 1). Following these changes,the sequence of helices from the N to C terminus of the new proteinwould be (alpha) A-B-C-E-F'-F-G-H-(beta)-C-D-E-F'-F-G-H-NEW-A-B. Theactual arrangement or the fusion regions would require careful design sothat new regions of structure did not extend into the dimer-dimerinterface region which is critical to cooperativity. Introduction ofamino acids containing basic or acidic residues into the molecule atcertain positions could allow some restoration of functionally importantsalt bridges and hydrogen bonds which could be lost as a result ofmanipulating the normal N and C termini. The above approach could alsoextend to the production of the entire hemoglobin molecule or individualdimers as single polypeptide chains, although in the latter case thiswould not be expected to offer stabilization against dimer formation.

For the purpose of providing the potential for disulfide bond formation,a cysteine may be introduced into either the α or β globin domain of theα₁ β₂ pseudodimer.

EXAMPLE 32 Production of Recombinant Hemoglobin in E. Coli

32.1 FERMENTATION

The two liter fermentation procedures described below can be used toobtain material for purification and functionality determinations.

To prepare the fermenter inoculum, 400 μl of seed stock are inoculatedinto 200 mL of 4.1 g/L KH₂ PO₄, 7.0 g/L KHPO₄, 2.0 g/L (NH₄)₂ SO₄, 1.0g/L Na₃ Citrate.2H₂ O, 154 mg/L MgSO₄.7H₂ O, up to 230 mg of proline,0.2% yeast extract, 1% glucose, 300 μl of 20 mg/ml thiamine insterile-filtered solution, 133 μl of 15 mg/ml tetracycline, and 0.6 mlof a trace metla solution. The trace metal solution contains 25 μg/mlFeCl₃ +6H₂ O, 1.3 μg/ml ZnCl₂, 2.0 μg/ml CoCl₂ +6H₂ O, 2 μg/ml Na₂ MoO₄+2H₂ O, 1.0 μg/ml CaCl₂ +2H₂ O, 2.54 μg/ml Cu(II)SO₄ +5H₂ O, 0.5 μg/mlH₃ BO₃, 1.2 μg/ml MnCl₂ +4H₂ O, and 100 μl/ml HCl dissolved in a 0.5MNa-citrate solution. This culture is allowed to grow at 37° C. on ashaker until an O.D. ₆₀₀ nm of 0.4-0.6 is achieved. The entire inoculumis then asceptically transferred to a 2-liter fermentor containing 2 g/LKH₂ PO₄, 3.6 g/L KHPO₄, 2.0 g/L (NH₄)₂ SO₄, 1 mL/L polypropyleneglycol-2000, 50 mL/L of 50% glucose, 100 mg/L of thiamine, 9.75 mg/L oftetracycline, 4 mL/L of trace metals, 1.54 g/L MgSO₄.7H₂ O and 3.68 g/LNa₃ Citrate.2H₂ O. The pH is maintained at 6.8 by addition of 15% to 30%NH₄ OH, dissolved oxygen is maintained at or above 30%, and 60% glucoseis added throughout the growth period, sufficient to maintain low butadequate levels of glucose in the culture (0.5 g/L-10 g/L). The cultureis grown between 25° and 30° C. to an OD₆₀₀ ˜10-40 prior to inductionwith 10-1000 μM IPTG. Upon induction of hemoglobin synthesis, the E.coli heme biosynthesis is supplemented by addition of hemin, either byaddition of the total mass of hemin required at induction, or byperiodic addition of hemin dissolved in 50 mM to 1M NaOH (e.g. one thirdof the total mass of hemin to be added to the fermentor is added atinduction, another third is added after 1/4 of the total time afterfermentation has elapsed, and the last third is added half-way throughthe induction period). Total hemin added can range from 50 to 300 mg/L.The fermenter is allowed to continue for 8-12 hours post-induction. Atthe end of this period, several 1 ml aliquots are removed from the brothfor determination of hemoglobin production.

32.2 CULTURE HARVEST BREAKAGE AND LYSATE PREPARATION

Cells can be harvested by centrifugation at 10,000×g for 10 minutes orthey can be collected by filtration by cross-flow filtration with 0.2 μmmembranes (e.g., Millipore Prostak). The cells are washed or resuspendedto 30% (w/v) in a 25 mM Na-borate/2 mM EDTA buffer (pH 9.3). Lysozyme(final conc. 0.02 g/L) and protease inhibitor (e.g., 1 mM benzamidine or50,000 I.U./L aprotonin) are added to the preparation. The suspendedcells are allowed to incubate for 30 minutes at 30°-31° C., and then arebroken by one or more passes through a homogenizer such as a GaulinModel 30-CD™ Homogenizer operated between 10 and 14 Kpsi or aMicrofluidics Corp. Cell Disruptor Microfluidizer™ set at 13 Kpsi. Theremainder of the process may be performed either under oxygen ornitrogen. The temperature of the lysate may be adjusted to 40° C. or thesolution may be utilized directly.

The lysed cells are then titrated to above pH .sup.˜ 6.8, preferablyabout 8.3, with 5N NaOH. Conductivity is adjusted to 30 Kmhos byaddition of NaCl. The broken cell extract is clarified and the celldebris washed with borate buffer containing protease inhibitor (asabove) by ultrafiltration. Since the hemoglobin product is soluble, itpasses through the filtration membranes.

After clarification all subsequent steps are performed in the cold (<10°C.). The solution may be purified by passage through either a strongcation exchange column (C), followed by a second strong cation exchangecolumn (C) followed by a strong anion exchange column (A), or by passagethrough C, then A then C, or alternatively, A, C, then A, or finallysimply by passage through only one strong cation exchange columnfollowed by A. Below we describe one possible purification scheme. TheCCA column purification order is described here.

The solution is oxygenated, then enough 10 mM sodium phosphate buffer isadded to the preparation to bring the conductivity down to ≧1800 mmhos.The resulting solution is then titrated to pH 6.7-7.0 with 0.5-5N H₃PO₄, loaded onto a strong cation exchange column (such as BioRadMacro-Prep 50 S column, IPF Biotechnics S-CPI or S-Spherodex columns,PerSeptive Biosystems Poros™ S or HS/MII columns, a PharmaciaS-Sepharose Fast Flow column, a TosoHaas Toyopearl™ SP-550C column, orWhatman SE52 or SE53 columns) pre-equilibrated with 10 mM sodiumphosphate, washed, and then eluted with 14-20 mM sodium phosphate bufferat pH 7.4-7.7. A fraction collector can be used to collect fractions ofinterest. Alternatively, the solution may be further processed asdescribed below.

A second cation exchange step can be performed. The solution from thefirst cation exchange step is diluted with an equal volume of deionizedwater, and the pH is adjusted to 6.8 with 5N phosphoric acid to provideadequate binding conditions for the second cation exchange column. Astrong cation exchanger such as those listed above is used. The columnis pre-equilibrated with 10 mM sodium phosphate, pH 6.8. After loading,the bound protein on the column is washed with approximately 2 columnvolumes of the equilibration buffer. A 14 mM sodium phosphate, pH 7.4wash, is then used to selectively elute the protein. During elution, thecolumn effluent is monitored for total protein by UV absorbance at 280nm.

Additional purification of recombinant hemoglobin can be achieved bymeans of an anion exchange column such as BioRad Macro-Prep 50 Q column,PerSeptive BioSystems Poros™ Q column, Pharmacia Q-Sepharose Fast Flowcolumn, TosoHaas Toyopearl™ QAE-550C column, or Whatman BioSystems QA52or DE53 column. Prior to use, the column is equilibrated with 20 mMtris-HCl buffer, pH 8.3. Material from the second cation exchangeelution is diluted or diafiltered with 20 mM tris-HCl buffer, pH 8.3 toallow binding to the chromatography resin. After loading, the column iswashed with 2 column volumes of equilibration buffer, and the proteineluted with 30 mM tris-HCl buffer, pH 7.2.

CONCENTRATION/BUFFER EXCHANGE

Protein from the final chromatography step can be concentrated andexchanged into 5 mM sodium phosphate/150 mM sodium chloride, pH 7.3 witha 30,000 molecular weight cutoff ultrafiltration membrane.

Reference Example A Reconstitution of Recombinant Alpha-Globin andRecombinant Beta-Globin with Heme and chemical Reduction to YieldArtificial Hemoglobin

Conventional methods of preparing artificial hemoglobin are exemplifiedby the following procedure.

The lyophilized recombinant alpha and beta-globins (100 mg each) wereindividually dissolved in 8M urea/50 mM Tris-Cl, pH 8.01/1 mM EDTA/1 mMDTT, diluted to a concentration of 5 mg/ml and incubated at roomtemperature for 3-4 hours. The alpha-globin was then diluted to 0.3mg/ml with chilled 20 mM K₂ HPO₄, pH 5.7/1 mM EDTA/1 mM DTT. Hemin (25mg) was dissolved in 2.4 mg 0.1M KOH, diluted with an equal volume of 1MKCN; this solution was then made 0.1 mg/ml in hemin and 20 mM K₂ HPO₄,pH 6.7 with stock phosphate buffer. Hemin from this solution was addedat a 2.8 molar excess to the chilled alpha-globin; and equal molaramount of beta-globin was added and the solution was dialyzed at 4° C.overnight against 0.1M K₂ HPO₄, pH 7.6/1 mM EDTA/1 mM KCN. Theartificial Hb solution was concentrated by ultra-filtration using aPM-10 membrane (Amicon) and transferred into a 200 ml screw-top testtube with a rubber septum. The hemoglobin solution was deoxygenated byevacuation and flushing with N₂, and then the solution was saturatedwith CO. 100 mM sodium dithionite solution was prepared anaerobically ina 20 ml screw-top test tube with rubber septum. 4.5 equivalents ofdithionite were added to the Hb solution with a syringe, and the mixtureincubated on ice for 15 min. The Hb solution was gel-filtered against 10mM Na phosphate buffer pH 6.0 on a 4×40 cm Sephadex G-25 (fine) column.The colored solution was then applied to a 2×10 cm-52 (Whatman) columnequilibrated with the same buffer and the chromatography was developedwith a linear gradient or 500 ml 10 mM Na phosphate buffer pH 6.0 and500 ml of 70 mM sodium phosphate buffer pH 6.9. CO was removed from Hbby photolysis under a stream of oxygen. Artificial Hgb prepared this wayis isolated in only about 25% yield from the fusion peptides but showsnative oxygen binding properties.

Reference Example B P₅₀ Determination

our preferred method of measuring P50 of purified hemoglobin solutionsfor the purpose of the appended claims is as follows:

Hemoglobin-oxygen equilibrium curves are measured using a HemoxAnalyzer™ (TCS Medical Products, Southampton, Pa.) at either 25° C. or37° C.+0.1° C. in 50 mM HEPES buffer/0.1M NaCl, pH 7.4. Oxygenequilibrium curves are measured by N2 deoxygenation of an oxyhemoglobinsolution that has been previously equilibrated with water-saturated O₂(for samples with a P50>10 torr) or with water-saturated compressed air.Absorbance readings at 568 and 558 nm are measured throughout the runfor determination of percent oxyhemoglobin in the sample. Precentoxyhemoglobin is directly proportional to log Aλ558/log Aλ568 and isindependent of path length. Both the absorbances and the oxygen pressureare sampled by a programmable-gain 12-bit analog-to-digital converter(Labmaster PGH, Scientific Solutions, Solon, Ohio) under computercontrol. The oxygen equilibrium curve is subjected to a low-apss digitalfilter. P₅₀ values (partial pressure of O₂ required for 50% saturationof oxygen binding sites) and Hill coefficients (^(n) max) are calculatedfrom the digitally filtered data by using software developed in ourlaboratory. The Hill coefficients are determined as the maximum slope ofthe functions dlog y/(1-y)!/dlog p, where y is % O₂ saturation and p ispartial pressure of O₂.

P50 may also be measured under other conditions, but it should be notedthat many environmental factors affect hemoglobin's oxygen affinity. Theeffect of pH, CO₂ inorganic anions, organic phosphates and temperatureon P50 are discussed in Bunn and Forget, HEMOGLOBIN: MOLECULAR, GENETICAND CLINICAL ASPECTS 37-47, 95-98 (W. B. Saunders Co., 1986).

Since many determinations of whole blood oxygen binding curves are madeunder standard physiologic conditions (37° C., pH, 7.4, PCO₂ 40 mm Hg),it may be necessary to adjust literature figures. In this context, itshould be noted that a 10° C. increase results in nearly a two-foldincrease in P50, while the dependence of P50 on pH is approximatelygiven as delta log P₅₀ /delta pH=-0.5.

Comparing P₅₀ values of purified Hb preparations to P₅₀ values of wholeblood can be problematic. Whole blood, or isolated RBC's contain manycomponents that naturally modulate the shape of the hemoglobin-oxygenbinding curve. The RBC encapsulates Hgb in the presence of a highconcentration of the effector molecule 2,3-DPG; a molecule that causesHgb to have a markedly lower affinity for O₂. Other intra-erythrocytecomponents also affect the shape of the binding curve: ATP, ClCO₂, H+,orthophosphate, methemoglobin and carboxyhemoglobin. The levels of thesesubstances may vary with age, sex and condition. These substances arenot normally present in purified HgB solutions and thus, the P₅₀ valueof purified Hgb is lower than that found in whole blood. One veryimportant modulator of Hgb-oxygen affinity is Cl- ion. Cl ion is foundoutside the erythrocyte in the blood serum at a physiologicconcentration of approximately 0.15M. Since Cl- causes a lower O₂affinity, a Hgb solution with a P₅₀ measured in vitro may well have muchlower O₂ affinity if infused into the blood stream. Another problem withmeasuring O₂ binding of whole blood is that RBCs are quite fragile andin the process of manipulating the erythrocyte into the instrument usedto measure the O₂ binding it is inevitable that at least a smallpercentage of the RBCs will lyse. Lysed RBCs leak Hgb into thesurrounding media away from 2,3-DPG; hence, since free Hgb has a higheraffinity than intraerythrocyte Hgb, lysed RBCs will have a higher O₂affinity and can cause a falsely low P50 value for whole blood P50determinations. It is widely accepted that under physiologic conditionswhile blood has a P50 value of 26-28 mm Hg. When Hgb is isolated fromwhole blood, however, the measured P50 is on the order of 1-10 mm Hgdepending on the investigator's experimental conditions. For thesereasons it is most accurate to measure Hgb-oxygen equilibria withpurified Hgb molecules under strict conditions of buffer, pH and saltconcentrations. Unfortunately, there are no accepted "standards" for allinvestigators to measure Hgb oxygen binding for in vitro systems.

Still, as many mutant hemoglobins are first identified in patient'swhole blood, one would like to be able to compare the relativeaffinities of native and mutant Hgb for O2 between whole blood andpurified Hgb preparations. An example of this is Hgb Chico (beta lys⁶⁶-thr). If one examined only the P50 value of the purified mutant Hgb(10.1 mmHg) one would note that Hgb has a P₅₀ value less than that fornormal whole blood (27.2 mmHg). Still, when that hemoglobin is measuredin RBCs under physiologic conditions it is apparent that it does have ahigher P₅₀ than normal whole blood (38 mmHg). One cannot predict thedegree that the P₅₀ value will change going from whole blood Chico to apurified Hgb Chico if it were infused into the bloodstream as a bloodsubstitute. One can conclude however, that the P₅₀ will be higher thanit is in pure form, and that by reacting the mutant Hgb with organicphosphates that P₅₀ so will be even higher.

                  TABLE 1                                                         ______________________________________                                        PRIMARY STRUCTURE OF HUMAN GLOBIN SUBUNITS                                    Helix α Zeta    Helix*                                                                              β                                                                              δ                                                                            Gamma  ε                       ______________________________________                                        NA1    1 Val  Ser     NA1    1 Val                                                                              Val  Gly    Val                                                   NA2    2 His                                                                              His  His    His                             NA2    2 Leu  Leu     NA3    3 Leu                                                                              Leu  Phe    Phe                             A1     3 Ser  Thr     A1     4 Thr                                                                              Thr  Thr    Thr                             A2     4 Pro  Lys     A2     5 Pro                                                                              Pro  G1u    Ala                             A3     5 Ala  Thr     A3     6 Glu                                                                              Glu  Glu    Glu                             A4     6 Asp  Glu     A4     7 Glu                                                                              Glu  Asp    Glu                             A5     7 Lys  Arg     A5     8 Lys                                                                              Lys  Lys    Lys                             A6     8 Thr  Thr     A6     9 Ser                                                                              Thr  Ala    Ala                             A7     9 Asn  Iie     A7     10 Ala                                                                             Ala  Thr    Ala                             A8     10 Val Ile     A8     11 Val                                                                             Val  Ile    Val                             A9     11 Lys Val     A9     12 Thr                                                                             Asn  Thr    Thr                             A1O    12 Al  Ser     A10    13 Ala                                                                             Ala  Ser    Ser                             A11    13 Ala Met     A11    14 Leu                                                                             Leu  Leu    Leu                             A12    14 Trp Trp     A12    15 Trp                                                                             Trp  Trp    Trp                             A13    15 Gly Ala     A13    16 Gly                                                                             Gly  Gly    Ser                             A14    16 Lys Lys     A14    17 Lys                                                                             Lys  Lys    Lys                             A15    17 Val Ile     A15    18 Val                                                                             Val  Val    Met                             A16    18 Gly Ser                                                             AB1    19 Ala Thr                                                             B1     20 His Gln     B1     19 Asn                                                                             Asn  Asn    Asn                             B2     21 Ala Ala     B2     20 Val                                                                             Val  Val    Val                             B3     22 Gly Asp     B3     21 Asp                                                                             Asp  Glu    Glu                             B4     23 Glu Thr     B4     22 Glu                                                                             Ala  Asp    Glu                             B5     24 Tyr Ile     B5     23 Val                                                                             Val  Ala    Ala                             B6     25 Gly Gly     B6     24 Gly                                                                             Gly  Gly    Gly                             B7     26 Ala Thr     B7     25 Gly                                                                             Gly  Gly    Gly                             B8     27 Glu Glu     B8     26 Glu                                                                             Glu  Glu    Glu                             B9     28 Ala Thr     B9     27 Ala                                                                             Ala  Thr    Ala                             B10    29 Leu Leu     B10    28 Leu                                                                             Leu  Leu    Leu                             B11    30 Glu Glu     B11    29 Gly                                                                             Gly  Gly    Gly                             B12    31 Arg Arg     B12    30 Arg                                                                             Arg  Arg    Arg                             B13    32 Met Leu     B13    31 Leu                                                                             Leu  Leu    Leu                             B14    33 Phe Phe     B14    32 Leu                                                                             Leu  Leu    Leu                             B15    34 Leu Leu     B15    33 Val                                                                             Val  Val    Val                             B16    35 Ser Ser     B16    34 Val                                                                             Val  Val    Val                             C1     36 Phe His     C1     35 Tyr                                                                             Tyr  Tyr    Tyr                             C2     37 Pro Pro     C2     36 Pro                                                                             Pro  Pro    Pro                             C3     38 Thr Gln     C3     37 Trp                                                                             Trp  Trp    Trp                             C4     39 Thr Thr     C4     38 Thr                                                                             Thr  Thr    Thr                             C5     40 Lys Lys     C5     39 Gln                                                                             Gln  Gln    Gln                             C6     41 Thr Thr     C6     40 Arg                                                                             Arg  Arg    Arg                             C7     42 Tyr Tyr     C7     41 Phe                                                                             Phe  Phe    Phe                             CE1    43 Phe Phe     CD1    42 Phe                                                                             Phe  Phe    Phe                             CE2    44 Pro Pro     CD2    43 Glu                                                                             Glu  Asp    Asp                             CE3    45 His His     CD3    44 Ser                                                                             Ser  Ser    Ser                             CE4    46 Phe Phe     CD4    45 Phe                                                                             Phe  Phe    Phe                                                   CD5    46 Gly                                                                             Gly  Gly    Gly                             CE5    47 Asp Asp     CD6    47 Asp                                                                             Asp  Asn    Asn                             CE6    48 Leu Leu     CD7    48 Leu                                                                             Leu  Leu    Leu                             CE7    49 Ser His     CD8    49 Ser                                                                             Ser  Ser    Ser                             CE8    50 His Pro     D1     50 Thr                                                                             Ser  Ser    Ser                                                   D2     51 Pro                                                                             Pro  Ala    Pro                                                   D3     52 Asp                                                                             Asp  Ser    Ser                                                   D4     53 Ala                                                                             Ala  Ala    Ala                                                   D5     54 Val                                                                             Val  Ile    Ile                                                   D6     55 Met                                                                             Met  Met    Leu                             CE9    51 Gly Gly     D7     56 Gly                                                                             Gly  Gly    Gly                             E1     52 Ser Ser     E1     57 Asn                                                                             Asn  Asn    Asn                             E2     53 Ala Ala     E2     58 Pro                                                                             Pro  Pro    Pro                             E3     54 Gln Gln     E3     59 Lys                                                                             Lys  Lys    Lys                             E4     55 Val Leu     E4     60 Val                                                                             Val  Val    Val                             E5     56 Lys Arg     E5     61 Lys                                                                             Lys  Lys    Lys                             E6     57 Gly Ala     E6     62 Ala                                                                             Ala  Ala    Ala                             E7     58 His His     E7     63 His                                                                             His  His    His                             E8     59 Gly Gly     E8     64 Gly                                                                             Gly  Gly    Gly                             E9     60 Lys Ser     E9     65 Lys                                                                             Lys  Lys    Lys                             E10    61 Lys Lys     E10    66 Lys                                                                             Lys  Lys    Lys                             E11    62 Val Val     E11    67 Val                                                                             Val  Val    Val                             E12    63 Ala Val     E12    68 Leu                                                                             Leu  Leu    Leu                             E13    64 Asp Ala     E13    69 Gly                                                                             Gly  Thr    Thr                             E14    65 Ala Ala     E14    70 Ala                                                                             Ala  Ser    Ser                             E15    66 Leu Val     E15    71 Phe                                                                             Phe  Leu    Phe                             E16    66 Thr Gly     E16    72 Ser                                                                             Ser  Gly    Gly                             E17    68 Asn Asp     E17    73 Asp                                                                             Asp  Asp    Asp                             E18    69 Ala Ala     E18    74 Gly                                                                             Gly  Ala    Ala                             E19    70 Val Val     E19    75 Leu                                                                             Leu  Ile, Thr                                                                             Ile                             E20    71 Ala Lys     E20    76 Ala                                                                             Ala  Lys    Lys                             EF1    72 His Ser     EF1    77 His                                                                             His  His    Asn                             EF2    73 Val Ile     EF2    78 Leu                                                                             Leu  Leu    Met                             EF3    74 Asp Asp     EF3    79 Asp                                                                             Asp  Asp    Asp                             EF4    75 Asp Asp     EF4    80 Asn                                                                             Asn  Asp    Asn                             EF5    76 Met Ile     EF5    81 Leu                                                                             Leu  Leu    Leu                             EF6    77 Pro Gly     EF6    82 Lys                                                                             Lys  Lys    Lys                             EF7    78 Asn Gly     EF7    83 Gly                                                                             Gly  Gly    Pro                             EF8    79 Ala Ala     EF8    84 Thr                                                                             Thr  Thr    Ala                             F1     80 Leu Leu     F1     85 Phe                                                                             Phe  Phe    Phe                             F2     81 Ser Ser     F2     86 Ala                                                                             Ser  Ala    Ala                             F3     82 Ala Lys     F3     87 Thr                                                                             Gln  Gln    Lys                             F4     83 Leu Leu     F4     88 Leu                                                                             Leu  Leu    Leu                             F5     84 Ser Ser     F5     89 Ser                                                                             Ser  Ser    Ser                             F6     85 Asp Glu     F6     90 Glu                                                                             Glu  Glu    Glu                             F7     86 Leu Leu     F7     91 Leu                                                                             Leu  Leu    Leu                             F8     87 His His     F8     92 His                                                                             His  His    His                             F9     88 Ala Ala     F9     93 Cys                                                                             Cys  Cys    Cys                             FG1    89 His Tyr     FG1    94 Asp                                                                             Asp  Asp    Asp                             FG2    90 Lys Ile     FG2    95 Lys                                                                             Lys  Lys    Lys                             FG3    91 Leu Leu     FG3    96 Leu                                                                             Leu  Leu    Leu                             FG4    92 Arg Arg     FG4    97 His                                                                             His  His    Hjs                             FG5    93 Val Val     FG5    98 Val                                                                             Val  Val    Val                             G1     94 Asp Asp     G1     99 Asp                                                                             Asp  Asp    Asp                             G2     95 Pro Pro     G2    100 Pro                                                                             Pro  Pro    Pro                             G3     96 Val Val     G3    101 Glu                                                                             Glu  Glu    Glu                             G4     97 Asn Asn     G4    102 Asn                                                                             Asn  Asn    Asn                             G5     98 Phe Phe     G5    103 Phe                                                                             Phe  Phe    Phe                             G6     99 Lys Lys     G6    104 Arg                                                                             Arg  Lys    Lys                             G7    100 Leu Leu     G7    105 Leu                                                                             Leu  Leu    Leu                             G8    101 Leu Leu     G8    106 Leu                                                                             Leu  Leu    Leu                             G9    102 Ser Ser     G9    107 Gly                                                                             Gly  Gly    Gly                             G10   103 His His     G10   108 Asn                                                                             Asn  Asn    Asn                             G11   104 Cys Cys     G11   109 Val                                                                             Val  Val    Val                             G12   105 Leu Leu     G12   110 Leu                                                                             Leu  Leu    Met                             G13   106 Leu Leu     G13   111 Val                                                                             Val  Val    Val                             G14   107 Val Val     G14   112 Cys                                                                             Cys  Thr    Ile                             G15   108 Thr Thr     G15   113 Val                                                                             Val  Val    Ile                             G16   109 Leu Leu     G16   114 Leu                                                                             Leu  Leu    Leu                             G17   110 Ala Ala     G17   115 Ala                                                                             Ala  Ala    Ala                             G18   111 Ala Ala     G18   116 His                                                                             Arg  Ile    Thr                             G19   112 His Arg     G19   117 His                                                                             Asn  His    His                             GH1   113 Leu Phe     GH1   118 Phe                                                                             Phe  Phe    Phe                             GH2   114 Pro Pro     GH2   119 Gly                                                                             Gly  Gly    Gly                             GH3   115 Ala Ala     GH3   120 Lys                                                                             Lys  Lys    Lys                             GH4   116 Glu Asp     GH4   121 Glu                                                                             Glu  Glu    Glu                             GH5   117 Phe Phe     GH5   122 Phe                                                                             Phe  Phe    Phe                             H1    118 Thr Thr     H1    123 Thr                                                                             Thr  Thr    Thr                             H2    119 Pro Ala     H2    124 Pro                                                                             Pro  Pro    Pro                             H3    120 Ala Glu     H3    125 Pro                                                                             Gln  Glu    Glu                             H4    121 Val Ala     H4    126 Val                                                                             Met  Val    Val                             H5    122 His His     H5    127 Gln                                                                             Gln  Gln    Gln                             H6    123 Ala Ala     H6    128 Ala                                                                             Ala  Ala    Ala                             H7    124 Ser Ala     H7    129 Ala                                                                             Ala  Ser    Ala                             H8    125 Leu Trp     H8    130 Tyr                                                                             Tyr  Trp    Trp                             H9    126 Asp Asp     H10   131 Gln                                                                             Gln  Gln    Gln                             10    127 Lys Lys     H10   132 Lys                                                                             Lys  Lys    Lys                             H11   128 Phe Phe     H11   133 Val                                                                             Val  Met    Leu                             H12   129 Leu Leu     H12   134 Val                                                                             Val  Val    Val                             H13   130 Ala Ser     H13   135 Ala                                                                             Ala  Thr    Ser                             H14   131 Ser Val     H14   136 Gly                                                                             Gly  Gly, Ala                                                                             Ala                             H15   132 Val Val     H15   137 Val                                                                             Val  Val    Val                             H16   133 Ser Ser     H16   138 Ala                                                                             Ala  Ala    Ala                             H17   134 Thr Ser     H17   139 Asn                                                                             Asn  Ser    Ile                             H18   135 Val Val     H18   140 Ala                                                                             Ala  Ala    Ala                             H19   136 Leu Leu     H19   141 Leu                                                                             Leu  Leu    Leu                             H20   137 Thr Thr     H20   142 Ala                                                                             Ala  Ser    Ala                             H21   138 Ser Glu     H21   143 His                                                                             His  Ser    His                             HC1   139 Lys Lys     HC1   144 Lys                                                                             Lys  Arg    Lys                             HC2   140 Tyr Tyr     HC2   145 Tyr                                                                             Tyr  Tyr    Tyr                             HC3   141 Arg Arg     HC3   146 His                                                                             His  His    His                             ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Oligonucleotide Sequences Used to Create Di-Alpha                             Globin Genes                                                                  ______________________________________                                        α.sup.1 (Arg)-Gly-Met(Leu)α.sub.2 Linker                          BstB1                             Eagl                                              CGAAATACCGTGGTATGCTGTCTCC                                                          TTTATGGCACCATACGACAGAGGCCGG                                        α.sup.1 (Arg)-Gly-Gly-(Val)α.sub.2 Linker                         BstB1                             Eagl                                              CGAAATACCGTGGTGGTGTTCTGTCTCC                                                       TTTATGGCACCACCACAAGACAGAGGCCGG                                     α.sup.1 (Arg)-Gly-(Val)α.sub.2 Linker                             BstBl                             Eagl                                              CGAAATACCGTGGTGTTCTGTCTCC                                                          TTTATGGCACCACAAGACAGAGGCCGG                                        α.sup.1 (Arg)-Gly-Gly-(Leu)α.sub.2                                BstBl                             Eagl                                              CGAAATACCGTGGTGGTCTGTCTCC                                                          TTTATGGCACCACCAGACAGAGGCCGG                                        α.sup.1 (Arg)-(Val)α.sub.2                                        BstBl                             Eagl                                              CGAAATACCGTGTTCTGTCTCC                                                             TTTATGGCACAAGACAGAGGCCGG                                           ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    Oligonucleotide Sequences to Create `Beta Globin Mutations                    __________________________________________________________________________    β.sup.67 val-->ile                                                       NcolKpnl                                                                      CATGGTAAAAAAatcCTGGGTGCTTTCTCTGACGGTCTGGCTCACCTGGACAACCTGAAAGGTAC                 CATTTTTTtagGACCCACGAAAGAGACTGCCAGACCGAGTGGACCTGTTGGACTTTC                 .sup.β67 val-->ile; .sup.β82 lys-->arg                              NcolKpnl                                                                      CATGGTAAAAAAatcCTGGGTGCTTTCTCTGACGGTCTGGCTCACCTGGACAACCTGcgtGGTAC                 CATTTTTTtagGACCCACGAAAAGAGACTGCCAGACCAGTGGACCTGTTGGACgcaC                 β.sup.102 asn-->thr                                                      SaclSpel                                                                          CCACTGCGACAAACTGCACGTTGACCCGGAAaccTTCCGTCTGCTGGGTAACGTA                   TCGAGGTGACGCTGTTTGACGTGCAACTGGGCCTTtggAAGGCAGACGACCCATTGCATGATC               __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                        P.sub.50 Values for Hemoglobin-Like Proteins (37° C.)                  Hemoglobin                                                                    ______________________________________                                        des-val Hgb          13.3                                                     dialpha (arg--gly--met) Hgb                                                                        9.0                                                      dialpha beta.sup.67 val-->ile Hgb                                                                  24.7                                                     dialpha (arg--gly--gly--val) Hgb                                                                   9.0                                                      ______________________________________                                    

                                      TABLE 5                                     __________________________________________________________________________    Synthetic Oligonucleotides Used For The Synthesis Of                          pGAP                                                                          __________________________________________________________________________    1 TCGACTGAAA AAAAAGGTTT AAACCAGTTC CCTGAAATTA TTCCCCTACT                        TGACTAATAA GTATATAAAG                                                       2 CAATACCTAC CGTTTATATA CTTATTAGTC AAGTAGGGGA ATAATTTCAG                        GGAACTGGTT TAAACCTTTT TTTTCAG                                               3 ACGGTAGGTA TTGATTGTAA TTCTGTAAAT CTATTTCTTA AACTTCTTGA                        ATTCTACTTT TATAGTTAGT CTTTTTTTTA GTTTT                                      4 AAGTTCTTGG TGTTTTAAAA CTAAAAAAAA GACTAACTAT AAAAGTAGAA                        AGAAGTTTAA GAAATAGATT TACAGAATTA CAAT                                       5 AAAACACCAA GAACTTAGTT TCGAATAAAC ACACATAAAT AAACCATGGT                        TAACT                                                                       6 CTAGAGTTAA CCATGGTTTA TTTATGTGTG TTTATTCGAA ACT                             __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Synthetic Oligonucleotides Used For The Synthesis                             Of The Galactose Upstream Activator                                           __________________________________________________________________________    1 CGTACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTC                           TCCTCCGTGCGTCCTCGTC TTCACCGGTCGC                                            2 AGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGGG                            CTTCTAATCCGTACGCATG                                                         3 GTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAAT                                AAAGATTCTACA#ACTAGCTTTT ATGGTTATGAAGAGGAAAAT                                4 ATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCG                            GCGCGAGGCACATCTGCGTT TCAGGAACGCGACCGGTGAAGAC                                5 TGGCAGTAACCTGGCCCCACAAACCTCAAATGAACGAAATCAAATTA                               ACAACCAGATATC                                                               6 TCGAGATATCTGGTTGTTAATTTGATTCGTTCATTTGAGGTTTGTGG                               GGCCAGGTTACTGCCAATTTTCCTCTTC                                                __________________________________________________________________________

                  TABLE 7                                                         ______________________________________                                        Codon Preferences in Yeast                                                    ______________________________________                                        Ala            GCU, GCC                                                       Ser            UCU, UCC                                                       Thr            ACU, ACC                                                       Val            GUU, GUC                                                       Ile            AUU, AUC                                                       Asp            GAC                                                            Phe            UUC                                                            Tyr            UAC                                                            Cys            UGU                                                            Asn            AAC                                                            His            CAC                                                            Arg            AGA                                                            Glu            GAA                                                            Leu            UUG                                                            Lys            AAG                                                            Gly            GGU                                                            Gln            CAA                                                            Pro            CCA                                                            Met            AUG (No alternative Codons)                                    Trp            UGG (No alternative Codons)                                    ______________________________________                                         Source: Bennetzen and Hall, J. Biol. Chem., 257:3026-31 (1982).          

                  TABLE 8                                                         ______________________________________                                        OLIGONUCLEOTIDES USED FOR THE CONSTRUCTION OF A                               GENE ENCODING A TANDEM ALPHA-GLOBIN DIMER.                                    ______________________________________                                        AL-1SS                                                                        5'-tgcacgcttctttggacaagttcttggcttctgtttctactgtgttaactagtaagt                  acagaggtggtgttttgtctcctgcagacaagactaac-3'                                     AL-2SS                                                                        5'-gttaaggctgcttggggtaaggttggtgctcacgctggtgaatacggtgctgaagcttt                ggaaaggatgttcttgtct-30'                                                       AL-1AS                                                                        5'-tgcaggagacaaaacaccacctctgtacttactagttaacacagtagaaacagaagcca                agaacttgtccaaagaagcg-3'                                                       AL-2AS                                                                        5'-ggaaagacaagaacatcctttccaaagcttcagcaccgtattcacccagcgtgagcacc                aaccttaccccaagcagccttaacgttagtcttgtc-3'                                       ______________________________________                                         Note: NcoIALPHA-1-ApaL1; FoK1ALPHA-2-Sal1; ApaLIRGGV-Fok1                

                  TABLE 9                                                         ______________________________________                                        Oxygen Affinity of Recombinant Mutant Hemoglobin                              Generated From FX-Hgb and Tryptic Digestion                                   ______________________________________                                        Oxygen affinity measured at 37° C. in 50 mM Bis-Tris                   (as Opposed to 50 mM HEPES buffer) pH 7.4, 0.1M NaCl on a                     Hemox-Analyzer. Solutions were 60 uM in heme and measured                     between 130 and 1.2 torr oxygen tension.                                      ______________________________________                                                        P.sub.50                                                      ______________________________________                                        HgB A.sub.o       9.5                                                         rHgB A.sub.o      9.2                                                         HgB Providence    10.2                                                        HgB Kansas        11.3                                                        HgB (beta.sup.67 val-->ile)                                                                     22.4                                                        ______________________________________                                    

The value for Hgb A₀ is of course for free hemoglobin in solution. TheP₅₀ of whole blood is much higher.

                  TABLE 10                                                        ______________________________________                                        Effects of NaCl and Inositol Hexaphosphate on Oxygen                          Binding to Hemoglobin A.sub.o and Recombinant des-Fx Hgb                      P.sub.50             P.sub.50                                                 0.1M NaCl       0M NaCl  2.2 mM IHP                                                                              0M 1HP                                     ______________________________________                                        Hgb A.sub.o                                                                           6.6         2.8      51.1    6.6                                      des-Fx Hgb                                                                            4.9         3.9      5.5     4.9                                      ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Distribution Of Fx-Alpha, FX-Beta and FX-Hgb in E. coli.                                    milligrams of protein                                                         per OD-liter of E. coli                                                       Soluble                                                                             Insoluble                                                 ______________________________________                                        FX-Alpha        36       0                                                    FX-Beta         21      21                                                    FX-Hgb          188      0                                                    ______________________________________                                    

                                      TABLE 100                                   __________________________________________________________________________    RFE                                                                              STRAIN                                                                             INDUCT TEMP                                                                           FINAL OD                                                                            MG DI-A + B                                                                          MG/OD-L                                                                            MG RHGB                                                                             RHGB MB/OD-L                          __________________________________________________________________________     1 BL21 30.0    6.6               0.0   0.0                                    5 B121HA                                                                             37.0    9.3               0.0   0.0                                    6 JM109                                                                              37.0    7.1   234.0  16.5 5.5   0.6                                    7 JML09                                                                              37.0    11.3  372.0  16.5 25.0  1.1                                    8 JM109                                                                              37.0    6.6   442.0  33.5 49.0  3.7                                   10 SCS1 37.0    15.2  754.0  24.8 38.0  1.2                                   11 JM109                                                                              25.0    11.5              26.0  1.1                                   12 JMl09                                                                              25.0    9.5               31.0  1.6                                   13 JM109                                                                              30.0    9.0               64.0  3.5                                   17 JM109                                                                              37.0    14.4              51.2  1.8                                   25 JM110                                                                              30.0    20.5              25.0  0.6                                   26 LE392                                                                              37.0    23.0  1040.0 22.6 4.1   0.1                                   27 JM110                                                                              30.0    27.0  334.0  6.2  47.0  0.9                                   30 JM110                                                                              37.0    23.6              16.0  0.3                                   31 23722                                                                              30.0    25.4              59.0  1.2                                   35 W3110                                                                              30.0    18.6              13.0  0.3                                   38 AG-1 30.0    13.0              57.0  2.1                                   49 DH-1 30.0    4.0               7.7   0.9                                   50 NM554                                                                              30.0    14.0              56.0  2.0                                   51 NM554                                                                              37.0    14.5              18.0  0.6                                   __________________________________________________________________________

                  TABLE 200                                                       ______________________________________                                        Bacterial and Yeast Vectors                                                   DEFINITIONS:                                                                  ______________________________________                                        ROP:         Gene which regulates plasid copy number                          ROP+:        Low copy number                                                  ROP-:        High copy number                                                 AR:          ampicillin resistance used for plasmid                                        selection                                                        TR:          tetracycline resistance used for plasmid                                      selection                                                        TS:          tetracycline sensitive, TR gene not                                           functional                                                       E4           tetracycline resistance used for plasmid                                      selection                                                        E5           tetracycline sensitive, TR gene not                                           functional                                                       FX-A:        FX-alpha globin gene                                             FX-B:        FX-beta globin gene                                              DFX-A:       Des-FX alpha globin gene                                         DFX-B:       Des-FX beta globin gene                                          DV-A:        Des-Val alpha globin gene                                        DV-B:        Des-Val beta globin gene                                         RV-Di-alpha: Di-alpha gene containing no amino acid                                        spacer (R = Arginine; V-Valine)                                  RGV-Di-alpha:                                                                              ADi-alpha globin gene containing a                                            single glycine (G) linker followed by a                                       valine                                                           RGM-Di-alpha:                                                                              Di-alpha globin gene containing a                                             single glycine (G) linker followed by a                                       methionine (M)                                                   RGGV-Di-alpha:                                                                             Di-alpha globin gene containing a two                                         glycine (G) linker followed by a                                              valine                                                           RGGGV-Di-alpha:                                                                            Di-alpha globin gene containing a                                             three glycine (G) linker followed by                                          a valine                                                         RPV-di-alpha Di-alpha gene fused by a single proline                                       (P) (yeast only)                                                 LACI:        gene encoding a represser which regulates                                     the TAC promoter                                                 LAC+:        Lac repressor gene on plasmid                                    LAC-:        no Lac repressor gene on plasmid                                 ______________________________________                                    

All bacterial plasmids listed below which contain alpha, di-alpha and/orbeta genes also have translational couplers. The pPL expression systemtranslationally couples the lambda N protein gene to a globin gene. Thedesignations "E4" and "E5" appear in the names of certain plasmids, andhave the meanings given above.

pKK223-3

Parental plasmid obtained commercially from Pharmacia LKB, 800Centennial Ave., P.O. Box 1327 Piscataway, N.J. 08855-1327; all otherplasmids derived from this parental plasmid. pKK223-3 has a TAC promoterfollowed by a poly-restriction site region to facilitate gene insertion.

AR, TS, ROP+, LAC-

1. pDL II-62m

pKK223-3 containing FX-A AR, TS, ROP+, LAC-

2. pDL II-10a

pKK223-2 containing FX-B AR, TS, ROP+, LAC-

3. PDL II-66A

Parental plasmids are (1) and (2); contains both FX-A and FX-B in singleoperon AR, TS, ROP+, LAC-

4. pGEM FX-A

Parental plasmids are (1) and pGem1 which is commerically available fromPromega Corporation, 2800 Woods Hollow Rd., Madison, Wis. 53711.

5. pGEM FX-B

pGEM containing FX-B, AR Parental plasmids are (2) and pGEM1

5a. pGEM di-alpha

Parentals are pGEM1 (see discussion of #4) and (2). The SmaI/PstIfragment of (29), containing the di-alpha gene, is excised and ligatedinto SmaI/PstI-cut pGEM1.

6. pDL II-83a

Parental is 4; contains DFX-A

7. pDL III-6f

Parental is 5; contains DFX-B, AR

8. pDL II-86c

Parentals are pKK223-3 and (6), contains DFX-A, AR, TS, ROP+, LAC-

9. pDL III-13e

Parentals are (7) and (8), pKK223-3 containing both DFX-A and DFX-B, AR,TS, ROP+, LAC-

10. pDL II-91f

Parental is (4) contains DV-A, AR

11. pDL II-95a

Parental is (7) contains DV-B, AR

12. pDL III-1a

Parentals are pKK223-3 and (10), contains DV-A AR, TS, ROP+, LAC-

13. pDL III-14c

Parentals are (11) and (12), contains DV-A and DV-B, AR, TS, ROP+, LAC-

13a. pDL III-38b

Parentals are (11) and (23) AR, TS, ROP+, LAC-

14. pDL III-47a

Parental is (13), contains RGM-di-alpha and DV-B, AR, TS, ROP+, LAC-

15. pDL III-82A

Parental is (13), contains RGGV-di-alpha and DV-B, AR, TS, ROP+, LAC-

16. pDL IV-8a

Parental is (13), contains RGV-di-alpha and DV-B, AR, TS, ROP+, LAC-

17. pDL IV-47b

Parental is (13), contains RV-di-alpha and DV-B AR, TS, ROP+, LAC-

18. pDL IV-66a

Parental is (13), contains RGGGV-di-alpha and DV-B, AR, TS, ROP+, LAC-

19. pDL IV-3a

Parental is (15), ROP gene is inactivated by insertion of a Not I linkerinto the PvuII site within the ROP gene AR, TS, ROP-, LAC-

20. pDL IV-38a

Parental is (15), contains the Nagai mutation in DV-B, AR, TS, ROP+,LAC-

21. pDL IV-58f

Parental is (20), ROP gene inactivated as in (19), AR, TS, ROP-, LAC-

22. pDL IV-59a

Parental is (21) and pBR322, which is commercially available from anumber of different suppliers. Contains a functional TR gene constructedin the following manner:

The EcoR1 site of pBR322 was changed to a BamH1 linker. This permittedthe removal of the 5' end 5' end of the TR gene from pBR322 as a BamH1fragment. This fragment was then inserted into the BamH1 site located atthe junction of the TAC promoter and the inactive TR gene of pKK223-3.Insertion of this fragment reactivates the TR gene if the fragmentinserts in the proper orientation. Selection of colonies on tetracyclineplates assures presence of the fragment in the proper orientation.

AR, TR, ROP-, LAC-

23. pJR V-83a

Parental is (11), contains DV-B with the Presbyterian mutation(asn108→lys), AR, TS, ROP+, LAC-

24. pJR VI-29a

Parentals are (15) and (23), contains RGGV-di-alpha and DV-B withPresbyterian mutation, AR, TS, ROP+, LAC-

25. pJR VI-53b

Parental is (24), made TR by insertion of BAMH1 fragment, AR, TR, ROP+,LAC-

26. pJR VI-61a

Parental is (25), made ROP-by insertion of Not I linker into PvuII siteAR, TR, ROP-, LAC-

27. pDL V-4a

Parentals are (16) and (26), contains RGV-di-alpha and DV-B withPresbyterian mutation AR, TS, ROP-, LAC-

28. pDL V-10a

Parental is (27), insertion of BamH1 fragment to convert to TR AR, TR,ROP-, LAC-

29. pDL V-16d (also named pSGE1.1E4 or simply pSGE1.1)

Parental is (28), contains LACI gene inserted into Not I site. LACI geneobtained using following protocol: Polymerase chain reaction (PCR)primers containing NOTI sites at their 5' ends were used to amplify theLACI gen. Following gel purification, the gene was inserted into the NOTI site of pDL V-1a AR, TR, ROP-, LAC+

Several other plasmid constructs have been designed to facilitate theincorporation of a second beta globin gene under regulation of its ownTAC promoter.

30. pDL IV-64a

Parental is (14), contains beta globin under regulation of a syntheticTAC promoter AR, TS, ROP+, LAC-

31. pDL IV-67a

Parental plasmids are (14) and (30), contains di-alpha under regulationof one pTAC, and DV-B under regulation of a second pTAC, DV-B isadjacent to di-alpha AR, TS, ROP-, LAC-

32. pJR VI-54a

Parental plasmids are (14) and (30), contains di-alpha and DV-B underregulation of one pTAC and a second DV-B under regulation of anotherpTAC. The second DV-B is inserted into the Pvu II site of the plasmidAR, TS, ROP-, LAC-

33. pPL Lambda,

Commercially available plasmid from Pharmacia LKB (see above); containspL promoter and coding region for N protein of lambda which can be usedfor expression of fusion or translationally coupled recombinantproteins.

34. pPL-alpha/beta

Parental plasmids are (13) and (33), contains DV-A and DV-B AR, ROP+

35. pPL-dialpha/beta

Parental is (34), contains RGV-di-alpha and DV-B AR, TS, ROP+, LAC-

36. pSGE0.1-LO

Parental plasmid is (35), ROP gene inactivated by insertion of NotIlinker into PvuII site in ROP gene AR, ROP-

37. pSGE0.1E4

Parentals are pSGE1.1E4 (29) and pDL II-95a (11), contains di-alphaglobin gene followed by wild type beta globin gene. The wild type betaglobin gene from pDL II-95a was excised with PstI and HindIII, gelpurified, and ligated into pSGE1.1E4 from which the beta Presbyteriangene had been removed with the same restriction enzymes.

38. pSGE0.0E4

Parentals are pSGE0.1E4 (37) and pDL II-91f (10), contains di-alphaglobin gene followed by wild type beta globin gene. The alpha globingene from pDL II-91f was excised with SmaI and PstI, gel purified, andligated into pSGE0.1E4 from which the di-alpha globin gene removed usingthe same restriction enzymes.

39. pPL lambda+TC

Parental is pPL lambda (33). pPL lambda was linearized with therestriction enzyme HpaI and synthetic oligonucleotides encoding thetranslational coupler region, and a portion of the 5' coding region ofalpha globin ligated into the HpaI site.

40. pSGE1.0E4

Parentals are pSGE1.1E4 (29) and pSGE0.0E4 (38), contains a single alphaglobin followed by beta globin gene containing the Presbyterianmutation. pSGE0.0E4 was digested with SmaI and PstI, the alpha globingene gel purified, and ligated into pSGE1.1E4 from which the di-alphagene had been removed with the same restriction enzymes.

41. pSGE223

Parental is commercially available pGEM-1. NheI restriction site hasbeen elimiated by digestion with NheI, T4 polymerase filing, andligation.

42. pSGE224

pSGE223 containing the beta Presbyterian gene.

Parentals are pSGE223 (41) and pSGE1.1E4 (29)

43. pSGE229

pSGE224 containing the 5 glycine di-beta linker sequences Parentalplasmid is pSGE224 (42) The linker sequences are synthetic DNA.

44. pSGE234

contains the di-beta globin gene. Parentals are pSGE224 (42) and pSGE229(43). The NheI fragment from pSGE229 contains the beta Presbyterian geneand the 5 glycine di-beta linker was ligated into NheI digested pSGE224.

45. pSGE1.1E5

Parental is pSGE1.1E4 (29). 5' end of the tet R gene was removed bydigestion with BamHI.

45a. pSGE0.0E5

Parental is pSGE0.0E4 (38). BamHI fragment encoding 5' end of tet R genehas been removed.

46. pSGE1.0E5

single alpha globin gene followed by beta Presbyterian gene. Parentalsare pSGE0.0E5 (45a) and pJRV-83a (23). The beta globin gene containingthe Presbyterian mutation was excised from (23) and ligated into (45a),from which the wild type beta globin gene had been removed with the sametwo restriction enzymes.

47. pSGE1.0E5

single alpha globin gene followed by 5 glycine linked di-beta gene.Parentals are pSGE234 (44) and pSGE1.0E5 (46). The PstI into HindIIIfragment from pSGE234 containing the di-beta gene was ligated inotpSGE1.0E5 from which the single beta globin gene had been removed usingthe same restriction enzymes.

48. pSGE1.05E4

Parentals are pSGE1.05E5 (47) and pSGE1.1E4 (29). The BamHI fragmentencoding the 5' end of the tet R gene from pSGE1.1E4 was ligated intothe BamHI site of pSGE1.05E5.

YEAST VECTORS

49. pSK+

Commercially available from Stratagene, LaJolla, Calif.

50. pGS2488

Derived from (49) by insertion of synthetic GAP 491 transcriptionalinitiation site.

51. pGS2888

Derived from (50) by conversion of KpnI site to SphI site.

52. pGS4788

Derived from (51) by insertion of synthetic GAL_(UAS) into (51) to formGALGAP hybrid promoter.

53. pLC IIFX-β-globin

Plasmid available from Kiyoshi Nagai, Medical Research Council, London,England; bears β globin gene

54. pUC19

Plasmid commercially available from Bethesda Research Laboratories,Gaithersburg, Md.

55. pSUC2-6Σ

Plasmid described by Stetler, et al., Biotechnology, 7:55-60 (1989)

56. pUC19β-globin

Derived from (53) and (54)

57. pGS1188

β-globin gene (from 56) is under control of the sigma promoter and theMFalpha terminator both from (55).

58. pGS3588

Derived from (57) and (52); sigma promoter replaced by GALGAP promoterof (52).

59. pGS3888

Derived from (58) by conversion of SmaI site to XhoI site.

60. pα-MRC

Plasmid available from K. Nagai, MRC; bears alpha-globin gene.

61. pGS4088

Derived from (59) by insertion of α-globin gene, replacing the β-globingene.

62. pSN(+)

Derived from (49) by changing KpnI site to NotI site.

63. pGS4888

Derived from (62) by insertion of pGGAP-α-globin expression cassettefrom (48).

64. pGS189

Derived from (63) by insertion of pGGAP-β-globin expression cassettefrom (47). Plasmid thus bears both GALGAP α globin and GALGAP β globingenes.

65. pC1U

Plasmid described by Stetler, et al.; Biotechnology, 7:55-60 (1980)

66. pC1N

Derived from (65) by addition of NotI site.

67. pGS289

Derived from (66) and (64) by insertion of α-globin and β-globinexpression cassettes.

68. pGS389

Same as (67), but with insert orientation reversed.

69. pYRp7

Plasmid described in Strathern, et al., The Molecular Biology of theYeast Saccharomyces (Cold Spring Harbor, 1981); source of TRP1 gene.

70. pC1T

Derived from (65) and (69) by replacing Ura3 gene (65) with Trp1 gene(69).

71. pGS4988

Derived from (70) by insertion of β-globin expression cassette from(59).

72. pGS4488

Derived from (65) by insertion of α-globin expression cassette from(61).

73. pGS4688

Same as (72), but with insert orientation reversed.

74. pGS4888

Derived from (62) by insertion of GGAP-alpha globin expression cassettefrom (61).

75. pGS1889

Derived from (74) by removal of PstI site.

76. pGS1989

Derived from (75) by removal of SpeI site.

77. pGS2189

Derived from (74) and (76); encodes di-alpha globin with RGGV linker.

78. pGS2989

Derived from (77) and (60), contains di-alpha globin gene and betaglobin gene.

79. pGS3089

Derived from (66) by insertion of expression cassette from (78).

80. Phagescript

Phage commercially available from Stratagene.

81. Phage Mpβ-globin Derived from (80) by insertion of GALGAP promoter.and β-globin gene from (78).

82. pGS3089 RGV desβ

Derived from (79) by deletion of XhoI fragment containing β-globinexpression cassette.

83. pGS3889

(79) with Presbyterian (βN108K) mutation.

84. pGS5189

(79) with Agenogi (βE90K) mutation.

85. pGS5689

(79) with Kansas (βN102T) mutation.

86. pGS4989

(79) with βV67I mutation.

87. pGS2989 RPV

(77) with RPV di-alpha linker

88. pGS2989 RGV

(77) with RGV di-alpha linker

89. pGS3089

(66) with Hgb expression cassette from (88)

90. pGS3089 RPV

(66) with Hgb expression cassette from (87)

                  TABLE 300                                                       ______________________________________                                        TABLE OF BACTERIAL STRAINS                                                           Strain    Availability                                                                            Expression                                         ______________________________________                                        1        BL21        Brookhaven                                                                              TAC                                            2        JM109       Commercial                                                                              TAC                                            3        SCS1        Commercial                                                                              TAC                                            4        JM110       ATCC      TAC                                            5        LE392       ATCC      TAC                                            6        23722       ATCC      TAC                                            7        W3110       ATCC      TAC                                            8        AG-1        Commercial                                                                              TAC                                            9        DH1         ATCC      TAC                                            10       NM554       Commercial                                                                              TAC                                            11       N99c1+      Commercial                                                                              PL                                             12       N4830-1     Commercial                                                                              PL                                             ______________________________________                                    

                  TABLE 400                                                       ______________________________________                                        HIGH OXYGEN AFFINITY, NATURALLY OCCURRING                                     HEMOGLOBIN MUTANTS                                                            Structure             Name                                                    ______________________________________                                        A. Alpha Chain Mutants                                                         6 (A4)       Asp->Ala    Sawara                                                            Asp->Asn    Dunn                                                              Asp->Val    Ferndown                                                          Asp->Tyr    Woodville                                                         Lys->Asn    Albany-Suma                                          40 (C5)      Lys->Glu    Kariya                                               44 (CE2)     Pro->Leu    Milledgeville                                                     Pro->Arg    Kawachi                                              45 (CE3)     His->Arg    Fort de France                                       85 (F6)      Asp->Asn    G-Norfolk                                            92 (FG4)     Arg->Gln    J-Cape Town                                                       Arg->Leu    Chesapeake                                           95 (G2)      Pro->Leu    G-Georgia                                                         Pro->Ser    Rampa                                                             Pro->Ala    Denmark Hill                                                      Pro->Arg    St. Luke's                                           97 (G4)      Asn->Lys    Dallas                                              126 (H9)      Asp->Asn    Tarrant                                             141 (HC3)     Arg->His    Suresnes                                                          Arg->Ser    J-Cubujuqui                                                       Arg->Leu    Legnano                                             B. Beta Chain Mutants                                                          2 (NA2)      His->Arg    Deer Lodge                                                        His->Gln    Okayama                                              20 (B2)      Val->Met    Olympia                                              23 (B5)      Val->Asp    Strasbourg                                                        Val->Phe    Palmerston North                                     34 (B16)     Val->Phe    Pitie-Salpetriere                                    36 (C2)      Pro->Thr    Linkoping                                            37 (C3)      Trp->Ser    Hirose                                               40 (C6)      Arg->Lys    Athens-Ga                                                         Arg->Ser    Austin                                               51 (D2)      Pro->Arg    Willamette                                                        Leu->His    Brisbane                                             79 (EF3)     Asp->Gly    G-Hsi-Tsou                                                        Lys->Thr    Rahere                                                            Lys->Met    Helsinki                                             89 (F5)      Ser->Asn    Creteil                                                           Ser->Arg    Vanderbilt                                           94 (FG1)     Asp->His    Barcelona                                                         Asp->Asn    Bunbury                                              96 (FG3)     Leu->Val    Regina                                               97 (FG4)     His->Gln    Malmo                                                             His->Leu    Wood                                                 99 (G1)      Asp->Asn    Kempsey                                                           Asp->His    Yakima                                                            Asp->Ala    Radcliffe                                                         Asp->Tyr    Ypsilanti                                                         Asp->Gly    Hotel-Dieu                                                        Asp->Val    Chemilly                                            100 (G2)      Pro->Leu    Brigham                                             101 (G3)      Glu->Lys    British Columbia                                                  Glu->Gly    Alberta                                                           Glu->Asp    Potomac                                             103 (G5)      Phe->Leu    Heathrow                                            109 (G11)     Val->Met    San Diego                                           121 (GH4)     Glu->Gln    D-Los Angeles                                                     Pro->Gln    Tu Gard                                                           Ala->Pro    Crete                                               140 (H18)     Ala->Thr    St.-Jacques                                         142 (H20)     Ala->Asp    Ohio                                                143 (H21)     His->Arg    Abruzzo                                                           His->Gln    Little Rock                                                       His->Pro    Syracuse                                            144 (HC1)     Lys->Asn    Andrew-Minneapolis                                  145 (HC2)     Tyr->His    Bethesda                                                          Tyr->Cys    Rainier                                                           Tyr->Asp    Fort Gordon                                                       Tyr->Term   McKees Rocks                                        146 (HC3)     His->Asp    Hiroshima                                                         His->Pro    York                                                              His->Leu    Cowtown                                             ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /label= AA- 1                                          /note= "Lys modified as Ac-lys"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 37                                                              (D) OTHER INFORMATION: /label= AA- 37                                         /note= "Leu is modified as Leu-amide"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       LysCysAlaGluLeuGluGlyArgLeuGluAlaLeuGluGlyArgLeu                              151015                                                                        GluAlaLeuGluGlyArgLeuGluAlaLeuGluGlyArgLeuGluAla                              202530                                                                        LeuGluGlyLysLeu                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyGluLeuGluGluLeuLeuLysLysLeuLysGluLeuLeuLysGly                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LeuArgArgGlnIleAspLeuGluValThrGlyLeu                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LysCysAlaGluLeuGluGlyLysLeuGluAlaLeuGluGlyLysLeu                              151015                                                                        GluAlaLeuGluGlyLysLeuGluAlaLeuGluGlyLysLeuGluAla                              202530                                                                        LeuGluGly                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCGAACGCGTTGTGCGCTCTGTCTGAT27                                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGTGCTCACGTTGATTGCATGCCGAACGCG30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTGACCAACGCTGTTTGCCACGTTGATGAT30                                              __________________________________________________________________________

We claim:
 1. A hemoglobin-like protein bearing prosthetic heme groupsand having reversible oxygen-binding activity, said protein comprisingat least one polypeptide which is a non-naturally occurring, geneticallyfused globin-like polypeptide comprising two or more globin-like domainsconnected by one or more peptide bonds into a single unbranchedpolypeptide chain, said chain being capable of associating with alphaand/or beta globin and incorporating heme to form a hemoglobin-likeprotein with reversible oxygen binding activity.
 2. The protein of claim1 said polypeptide comprising only two globin-like domains.
 3. Theprotein of claim 2 said protein being a di-alpha globin-like polypeptidecomprising first and second alpha globin-like polypeptide domains, saidchain being capable of associating with beta globin.
 4. The protein ofclaim 2, said protein being a di-beta globin-like polypeptide comprisingfirst and second beta-globin-like polypeptide domains, said chain beingcapable of associating with alpha-globin activity.
 5. The protein ofclaim 2, said protein being a pseudodimeric globin-like polypeptidecomprising an alpha globin-like polypeptide domain and a betaglobin-like polypeptide domain.
 6. The protein of claim 1 wherein insaid polypeptide a first and a second globin-like polypeptide domain areconnected by a peptide linker of one or more amino acids.
 7. The proteinof claim 6 wherein the amino acids of the linker in said polypeptide areselected from the group consisting of lysine, aspartic acid, arginine,serine, asparagine, proline and glycine.
 8. The protein of claim 7wherein the linker in said polypeptide consists of one or more glycines.9. The protein of claim 1 wherein in said polypeptide at least oneglobin-like domain has at least 75% sequence identity with human alphaglobin or human beta globin.
 10. The protein of claim 1 wherein in saidpolypeptide each globin-like domain is identical in sequence with humanalpha globin or human beta globin.
 11. The protein of claim 1 wherein ata predetermined position, a first globin-like domain has a cysteineresidue and a second globin-like domain has a residue other thancysteine.
 12. The protein of claim 11 wherein one of said predeterminedpositions is(a) a position in the alpha globin-like domain correspondingto a human alpha globin position selected from the group consisting ofhis72, asn78, asn68, ala71, thr67, lys7, lys11, thr8, ala12, thr118,lys16, ala45, glu116, gly15, his112, thr24, glu23, lys60, lys56, his50,gly51, glu53, ser49, asp47, gln54, his45, lys90, ala82, lys61, ala19,his20, asp85, ser81, asp75, asp74, lys139, asp64, and gly18, or (b) aposition in a beta globin-like domain corresponding to a human betaglobin position selected from the group consisting of asp79, his2, leu3,thr4, glu6, ser9, thr12, ala13, gly16, lys17, val18, asn19, val20,asp21, glu22, lys65, ser72, ala76, his77, asp79, asn80, gly83, ala86,thr87, glu90, lys95, lys59, glu43, ser44, asp47, ser49, thr50, ala53,asp52, lys61, glu121, lys120, thr123, lys66, asp73, ala62, his116, andhis117.
 13. The protein of claim 1, said protein being a pseudotetramer.14. The protein of claim 1, said protein being a pseudooctamer composedof two pseudotetramers, or of one pseudotetramer and one tetramer. 15.The protein of claim 14, said protein being(a) a polypeptide comprisingfour alpha globin-like domains, and, in the form of one or moreadditional polypeptides, four associable beta globin-like domains, or(b) a polypeptide comprising four beta globin-like domains, and, in theform of one or more additional polypeptides, four associable alphaglobin-like domains.
 16. The protein of claim 15, wherein the associabledomains are domains of pseudodimeric globin-like polypeptides.
 17. Theprotein of claim 15 wherein the associable domains are each provided asa separate polypeptide chain.
 18. A human hemoglobin-like proteinaccording to claim 1, said protein comprising:(a) a di-alpha globin-likepolypeptide and two beta-globin-like polypeptides; (b) adi-beta-globin-like polypeptide and two alpha globin-like polypeptides;(c) a di-alpha globin-like polypeptide and a di-beta globin-likepolypeptide; (d) an alpha/beta di-globin-like polypeptide, analpha-globin like polypeptide, and a beta globin-like polypeptide, or;(e) two alpha/beta digloblin-like poly-peptides.
 19. The protein ofclaim 18, said protein being a pseudotetramer.
 20. The protein of claim19, said protein comprising a di-alpha-globin-like polypeptide and twobeta-globin-like polypeptides.
 21. The protein of claim 20, wherein thealpha globin-like domains of said di-alpha-globin-like polypeptide eachhave at least 75% sequence identity with human alpha globin, and thebeta globin-like polypeptides each have at least 75% sequence identitywith human beta globin.
 22. The protein of claim 21, wherein the alphaglobin-like domains are identical in sequence with human alpha globinand the beta globin-like polypeptides are identical in sequence withhuman beta globin.
 23. The protein of claim 21 which is a low affinityhemoglobin mutant.
 24. The protein of claim 21 which is a high affinityhemoglobin mutant.
 25. The protein of claim 21 wherein the betaglobin-like polypeptide comprises a Presbyterian mutation.
 26. Theprotein of claim 18, said protein being a pseudooctamer composed of twopseudotetramers, or of one pseudotetramer and one tetramer.
 27. Theprotein of claim 26, said protein comprising two covalently bondedpseudotetramers, each pseudotetramer comprising (a) a pseudodimerselected from the group consisting of a di-alpha-globin-likepolypeptide, (b) a di-beta-globin-like polypeptide, and (c) analpha/beta globin-like polypeptide, the pseudodimers of eachpseudotetramer being covalently bonded, said pseudodimers beingasymmetric, said asymmetry being the presence at a amino acid positionof a cysteine in a first domain but not in a second domain.
 28. Theprotein of claim 27 wherein said pseudodimers are covalently bonded by adisulfide bridge between a cysteine of one psuedodimer and a cysteine ofthe other psuedodimer.
 29. The protein of claim 27 wherein saidpseudodimers are covalently bonded by a bridging moiety, other than adisulfide bridge, between a cysteine of one pseudodimer and a cysteineof the other pseudodimer.
 30. The protein of claim 29 wherein thebridging moiety is nonreducible under physiologic conditions.
 31. Theprotein of claim 29 wherein the bridging moiety increases theintravascular retention time of the protein.
 32. The protein of claim 29wherein the bridging moiety reduces the isoelectric point of theprotein.
 33. The protein of claim 26, said protein comprising a singleunbranched polypeptide comprising more than two alpha globin-likedomains or more than two beta globin-like domains.
 34. The protein ofclaim 18, said protein comprising more than two pseudotetramers.
 35. Theprotein of claim 34, each pseudotetramer comprising (a) a pseudodimerselected from the group consisting of a di-alpha-globin-likepolypeptide, (b) a di-beta-globin-like polypeptide, and (c) analpha/beta globin-like polypeptide, the pseudodimers of eachpseudotetramer being covalently bonded, said pseudodimers beingasymmetric, said asymmetry being the presence at a amino acid positionof a cysteine in a first domain but not in a second domain.
 36. Theprotein of claim 35, said pseudotetramers being covalently bonded to acoiled coil linker means.
 37. The protein of claim 35, saidpsuedotetramers being covalently bonded to a multihelical bundle linkermeans.
 38. The protein of claim 1 wherein said hemoglobin-like proteinis a low oxygen affinity mutant hemoglobin.
 39. The protein of claim 38wherein at least one of said low affinity mutations is(a) a mutation ofan alpha-globin mutation selected from the group consisting of 43phe→leu, 43 phe→val, 86 leu→arg, 94 asp→asn, 46 phe→thr, 46 phe→ser, 46phe→ala, 58 his→phe, 58 his→trp, 61 lys→thr, 61 lys→ser, 61 lys→met, 61lys→asn, 62 val→leu, 62 val→ile, 62 val→phe, 62 val→trp, 65 ala→asp, 94asp→gln, 94 asp→thr, 94 asp→ser, 94 asp→lys, 94 asp→gly, and 94 asp→arg,or (b) a mutation of a beta globin-like domain which corresponds to ahuman beta globin mutation selected from the group consisting of 1val→acetyl ala, 21 asp→gly, 24 gly→asp, 37 trp→arg, 38 thr→pro, 42phe→ser, 42 phe→leu, 42 phe→val, 45 phe→ser, 48 leu→arg, 61 lys→met, 65lys→gln, 66 lys→thr, 67 val→asp, 70 ala→asp, 73 asp→tyr, 73 asp→asn, 73asp→val, 82 lys→thr, 83 gly→asp, 90 glu→gly, 90 glu→lys, 91 leu→arg, 102asn→thr, 102 asn→ser, 102 asn→tyr, 102 asn→lys, 107 gly→arg, 108asn→asp, 108 asn→lys, 111 val→phe, 113 val→glu, 136 gly→asp, 21 asp→ala,21 asp→ser, 45 phe→ala, 45 phe→thr, 45 phe→val, 63 his→phe, 63 his→trp,66 lys→ser, 66 lys→asn, 67 val→phe, 67 val→trp, 67 val→ile, 70 ala→glu,70 ala→ser, 70 ala→thr, 96 leu→phe, 96 leu→his, 96 leu→lys, 98 val→trp,98 val→phe, 102 asn→asp, 102 asn→glu, 102 asn→arg, 102 asn→his, 102asn→gly, 108 asn→arg, and 108 asn→glu.
 40. The protein of claim 39,wherein at least one beta globin-like domain contains a 108 asn→lysmutation.
 41. The protein of claim 1, wherein said hemoglobin-likeprotein is a high oxygen affinity mutant hemoglobin.
 42. The protein ofclaim 41 wherein at least one of said high affinity mutations is(a) amutation of an alpha globin-like domain which corresponds to a humanalpha globin mutation selected from the group consisting of 6 Asp→Ala, 6Asp→Asn, 6 Asp→Val, 6 Asp→Tyr, 6 Lys→Asn, 40 Lys→Glu, 44 Pro→Leu, 44Pro→Arg, 45 His→Arg, 85 Asp→Asn, 92 Arg→Gln, 92 Arg→Leu, 95 Pro→Leu, 95Pro→Ser, 95 Pro→Ala, 95 Pro→Arg, 97 Asn→Lys, 126 Asp→Asn, 141 Arg→His,141 Arg→Ser, and 141 Arg→Leu, or (b) a mutation of a beta globin-likedomain which corresponds to a human beta globin mutation selected fromthe group consisting of 2 His→Arg, 20 Val→Met, 23 Val→Asp, 23 Val→Phe,34 Val→Phe, 36 Pro→Thr, 37 Trp→Ser, 40 Arg→Lys, 40 Arg→Ser, 51 Pro→Arg,51 Leu→His, 79 Asp→Gly, 79 Lys→Thr, 79 Lys→Met, 82 Lys→Asn, 89 Ser→Asn,89 Ser→Arg, 94 Asp→His, 94 Asp→Asn, 96 Leu→Val, 97 His→Gln, 97 His→Leu,99 Asp→Asn, 99 Asp→His, 99 Asp→Ala, 99 Asp→Tyr, 99 Asp→Gly, 99 Asp→Val,100 Pro→Leu, 101 Glu→Lys, 101 Glu→Gly, 101 Glu→Asp, 103 Phe→Leu, 109Val→Met, 121 Glu→Gln, 121 Pro→Gln, 121 Ala→Pro, 140 Ala→Thr, 142Ala→Asp, 143 His→Arg, 143 His→Gln, 143 His→Pro, 144 Lys→Asn, 145Tyr→His, 145 Tyr→Cys, 145 Tyr→Asp, 145 Tyr→Term, 146 His→Asp, 146His→Pro, and 146 His→Leu.
 43. The protein of claim 42 in which at leastone of said high affinity mutations is the Providence mutation beta 82Lys→Asn.
 44. The protein of claim 1 wherein each globin-like domain hasat least 75% sequence identity with human alpha globin or human betaglobin.
 45. The polypeptide of claim 44 wherein said globin-like domainsof said polypeptide are identical in sequence, except that at one ormore predetermined positions, a first globin-like domain has a cysteineresidue and a second globin-like domain has a residue other thancysteine.
 46. The protein of claim 1 wherein each alpha globin-likedomain of said genetically fused polypeptide is identical to human alphaglobin save for(a) one or more an oxygen affinity-modifyingmodifications characterized as(i) one or more low affinity mutations,selected from the group consisting of 43 phe→leu, 43 phe→val, 86leu→arg, 94 asp→asn, 46 phe→thr, 46 phe→ser, 46 phe→ala, 58 his→phe, 58his→trp, 61 lys→thr, 61 lys→ser, 61 lys→met, 61 lys→asn, 62 val→leu, 62val→ile, 62 val→phe, 62 val→trp, 65 ala→asp, 94 asp→gln, 94 asp→thr, 94asp→ser, 94 asp→lys, 94 asp→gly, and 94 asp→arg, or (ii) one or morehigh affinity mutations selected from the group consisting of 6 Asp→Ala,6 Asp→Asn, 6 Asp→Val, 6 Asp→Tyr, 6 Lys→Asn, 40 Lys→Glu, 44 Pro→Leu, 44Pro→Arg, 45 His→Arg, 85 Asp→Asn, 92 Arg→Gln, 92 Arg→Leu, 95 Pro→Leu, 95Pro→Ser, 95 Pro→Ala, 95 Pro→Arg, 97 Asn→Lys, 126 Asp→Asn, 141 Arg→His,141 Arg→Ser, and 141 Arg→Leu; and/or (b) a Cys substitution at one ormore sites selected from the group consisting of: his72, asn78, asn68,ala71, thr67, lys7, lys11, thr8, ala12, thr118, lys16, ala45, glu116,gly15, his112, thr24, glu23, lys60, lys56, his50, gly51, glu53, ser49,asp47,gln54, his45, lys90, ala82, lys61, ala19, his20, asp85, ser81,asp75, asp74, lys139, asp64, and gly18.
 47. The protein of claim 1wherein the beta globin-like domain of said genetically fusedpolypeptide is identical to human beta globin save for(a) one or moreoxygen affinity-modifying modifications characterized as(i) one or morelow affinity mutations selected from the group consisting of, 1val→acetyl ala, 21 asp→gly, 24 gly→asp, 37 trp→arg, 38 thr→pro, 42phe→ser, 42 phe→leu, 42 phe→val, 45 phe→ser, 48 leu→arg, 61 lys→met, 65lys→gln, 66 lys→thr, 67 val→asp, 70 ala→asp, 73 asp→tyr, 73 asp→asn, 73asp→val, 82 lys→thr, 83 gly→asp, 90 glu→gly, 90 glu→lys, 91 leu→arg, 102asn→thr, 102 asn→ser, 102 asn→tyr, 102 asn→lys, 107 gly→arg, 108asn→asp, 108 asn→lys, 111 val→phe, 113 val→glu, 136 gly→asp, 21 asp→ala,21 asp→ser, 45 phe→ala, 45 phe→thr, 45 phe→val, 63 his→phe, 63 his→trp,66 lys→ser, 66 lys→asn, 67 val→phe, 67 val→trp, 67 val→ile, 70 ala→glu,70 ala→ser, 70 ala→thr, 96 leu→phe, 96 leu→his, 96 leu→lys, 98 val→trp,98 val→phe, 102 asn→asp, 102 asn→glu, 102 asn→arg, 102 asn→his, 102asn→gly, 108 asn→arg, and 108 asn→glu, or (ii) one or more high affinitymutations selected from the group consisting of, 2 His→Arg, 20 Val→Met,23 Val→Asp, 23 Val→Phe, 34 Val→Phe, 36 Pro→Thr, 37 Trp→Ser, 40 Arg→Lys,40 Arg→Ser, 51 Pro→Arg, 51 Leu→His, 79 Asp→Gly, 79 Lys→Thr, 79 Lys→Met,82 Lys→Asn, 89 Ser→Asn, 89 Ser→Arg, 94 Asp→His, 94 Asp→Asn, 96 Leu→Val,97 His→Gln, 97 His→Leu, 99 Asp→Asn, 99 Asp→His, 99 Asp→Ala, 99 Asp→Tyr,99 Asp→Gly, 99 Asp→Val, 100 Pro→Leu, 101 Glu→Lys, 101 Glu→Gly, 101Glu→Asp, 103 Phe→Leu, 109 Val→Met, 121 Glu→Gln, 121 Pro→Gln, 121Ala→Pro, 140 Ala→Thr, 142 Ala→Asp, 143 His→Arg, 143 His→Gln, 143His→Pro, 144 Lys→Asn, 145 Tyr→His, 145 Tyr→Cys, 145 Tyr→Asp, 145Tyr→Term, 146 His→Asp, 146 His→Pro, and 146 His→Leu; and/or (b) a Cyssubstitution at one or more sites selected from the group consisting of:asp79, his2, leu3, thr4, glu6, ser9, thr12, ala13, gly16, lys17, val18,asn19, val20, asp21, glu22, lys65, ser72, ala76, his77, asp79, asn80,gly83, ala86, thr87, glu90, lys95, lys59, glu43, ser44, asp47, ser49,thr50, ala53, asp52, lys61, glu121, lys120, thr123, lys66, asp73, ala62,his116, and his117.
 48. The protein of claim 1, said protein comprisingmore than two pseudotetramers.
 49. An octameric hemoglobin-like protein,wherein (a) the two beta-globin-like polypeptides in the beta₁ positionare disulfide bonded and the two beta-globin-like polypeptides in thebeta₂ position are disulfide bonded, or (b) the two alpha-globin-likepolypeptides in the alpha₁ position are disulfide bonded and the twoalpha-globin-like polypeptides in the alpha₂ position are disulfidebonded.
 50. A genetically fused hemoglobin-like protein composed offewer than four polypeptide chains and having a half-life in thecirculatory system substantially in excess of that for des-Valhemoglobin.